EP3326649B1 - Anti-il-23p19-antikörper - Google Patents

Anti-il-23p19-antikörper Download PDF

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EP3326649B1
EP3326649B1 EP17208896.5A EP17208896A EP3326649B1 EP 3326649 B1 EP3326649 B1 EP 3326649B1 EP 17208896 A EP17208896 A EP 17208896A EP 3326649 B1 EP3326649 B1 EP 3326649B1
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antibody
seq
amino acid
mouse
humanized
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EP3326649A1 (de
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Gerald Henry NABOZNY
Annette Bettina GALLER
Patrick Garidel
William Troy LOGING
Steven John PADULA
Torsten Schultz-Fademrecht
Elaine Ee-Ling WANG
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Boehringer Ingelheim International GmbH
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Boehringer Ingelheim International GmbH
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Priority to EP22154240.0A priority Critical patent/EP4039275A1/de
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    • C07—ORGANIC CHEMISTRY
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    • C07K16/18—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans
    • C07K16/24—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans against cytokines, lymphokines or interferons
    • C07K16/244—Interleukins [IL]
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    • C07K2317/20—Immunoglobulins specific features characterized by taxonomic origin
    • C07K2317/24—Immunoglobulins specific features characterized by taxonomic origin containing regions, domains or residues from different species, e.g. chimeric, humanized or veneered
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    • C07K2317/30—Immunoglobulins specific features characterized by aspects of specificity or valency
    • C07K2317/34—Identification of a linear epitope shorter than 20 amino acid residues or of a conformational epitope defined by amino acid residues
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    • C07K2317/71—Decreased effector function due to an Fc-modification
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    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2317/00—Immunoglobulins specific features
    • C07K2317/90—Immunoglobulins specific features characterized by (pharmaco)kinetic aspects or by stability of the immunoglobulin
    • C07K2317/92—Affinity (KD), association rate (Ka), dissociation rate (Kd) or EC50 value
    • Y—GENERAL TAGGING OF NEW TECHNOLOGICAL DEVELOPMENTS; GENERAL TAGGING OF CROSS-SECTIONAL TECHNOLOGIES SPANNING OVER SEVERAL SECTIONS OF THE IPC; TECHNICAL SUBJECTS COVERED BY FORMER USPC CROSS-REFERENCE ART COLLECTIONS [XRACs] AND DIGESTS
    • Y02—TECHNOLOGIES OR APPLICATIONS FOR MITIGATION OR ADAPTATION AGAINST CLIMATE CHANGE
    • Y02A—TECHNOLOGIES FOR ADAPTATION TO CLIMATE CHANGE
    • Y02A50/00—TECHNOLOGIES FOR ADAPTATION TO CLIMATE CHANGE in human health protection, e.g. against extreme weather
    • Y02A50/30—Against vector-borne diseases, e.g. mosquito-borne, fly-borne, tick-borne or waterborne diseases whose impact is exacerbated by climate change

Definitions

  • This invention generally relates to anti-IL-23p19 antibodies for therapeutic use. More specifically, humanized anti-IL-23p19 antibodies and methods of use for the treatment of various diseases or disorders are disclosed. Pharmaceutical compositions comprising such compounds are also disclosed.
  • IL-12 a heterodimeric cytokine consisting of a p40 and a p35 protein subunit, has long been considered the hallmark cytokine of the innate immune response with major influence on adaptive immunity.
  • mice were resistant to Collagen Induced Arthritis (CIA) and Experimental Autoimmune Encephalomyelitis (EAE), p35-deficient mice were susceptible to both and even displayed exacerbated disease.
  • CIA Collagen Induced Arthritis
  • EAE Experimental Autoimmune Encephalomyelitis
  • IL-23 is composed of a common subunit (p40) with IL-12 and a unique p19 subunit. Despite this shared p40 subunit, the roles for IL-23 and IL-12 are quite different. IL-12 is important for Th1 responses via promotion of Th1 cell differentiation, proliferation and activation. In contrast, IL-23 supports the development and maintenance of a recently defined set of CD4 + T helper cells termed Th17 cells due to their ability to produce IL-17 and related cytokines. There is mounting evidence that IL-23 is involved in chronic autoimmune inflammation and the modulation of IL-23 activity could provide promising therapies against autoimmune diseases.
  • WO 2011/066369 discloses anti-IL-6 antibodies but mentions formulations thereof only in a general manner. This is also true for WO 2007/024846 .
  • anti-HER-2 and anti-DR5 antibodies are disclosed which are formulated in histidine-acetate buffer.
  • the aim of the present invention is to provide pharmaceutical compositions for antibody molecules, in particular for anti-IL23p19 antibodies.
  • a particular aim of the present invention is to provide pharmaceutical compositions for antibody molecules with favorable stability and storability.
  • the present invention addresses the above needs and provides pharmaceutical compositions comprising an antibody that binds to the p19 subunit of the IL-23 as defined in the claims.
  • the antibody of the compositions of the present invention binds to human IL-23 with high affinity and inhibits the IL-23 stimulated production of IL-17 from mouse splenocytes.
  • the antibody does not bind to nor antagonize IL-12, which is a closely related family member to IL-23.
  • anti-IL-23p19 antibodies that are derived from mouse hybridomas, for example monoclonal antibodies.
  • full length anti-IL-23p19 antibodies are provided.
  • anti-lL-23p19 humanized antibodies for example humanized monoclonal anti-IL-23p19 antibodies, for example full length humanized monoclonal anti-IL-23p19 antibodies.
  • a humanized antibody binds to human IL-23 with high affinity.
  • a humanized antibody also binds to cynomolgus IL-23 with high affinity.
  • a humanized antibody inhibits IL-23-induced STAT3 phosphorylation in DB cells.
  • a humanized antibody antagonizes the action of IL-23 by binding to the p19 subunit of IL-23, for example as measured by the inhibition of cytokines such as IL-17 and IL-22, whose production is stimulated by IL-23.
  • a humanized antibody has a favorable pharmacokinetic (PK) profile.
  • PK pharmacokinetic
  • a humanized antibody of the present disclosure has favorable biophysical properties, such as quality, stability or solubility, for example as defined by the percentage of antibody in monomer form.
  • Further reference aspects encompass DNA molecules encoding antibodies of the disclosure , expression vectors and host cells comprising such DNA molecules, and methods of making antibodies of the present invention.
  • the present invention further provides therapeutic uses for the antibodies in the pharmaceutical compositions of the present invention, in particular against immunological and autoimmune diseases.
  • the specification further provides an anti-IL-23p19 antibody or antigen-binding fragment thereof comprising a light chain CDR1 (L-CDR1) sequence of SEQ ID NO:1, 4, 6, 7, 8, 11, 15, 18, 19, 22, 27 or 30; a light chain CDR2 (L-CDR2) sequence of SEQ ID NO:2, 5, 9, 12, 16, 20, 23, 25, 28 or 31; a light chain CDR3 (L-CDR3) sequence of SEQ ID NO:3, 10, 13, 14, 17, 21, 24, 26, 29, or 32; a heavy chain CDR1 (H-CDR1) sequence of SEQ ID NO:33, 36, 38, 40, 43, 45, 48, 51, 54, 57, 60, 63, 66, 67, 68, 69, 77 or 80; a heavy chain CDR2 (H-CDR2) sequence of SEQ ID NO:34, 39, 41, 46, 49, 52, 55, 58, 61, 64, 70, 72, 73, 75, 78 or 81; and a heavy chain CDR3 (H
  • the anti-IL-23p19 antibody or antigen-binding fragment thereof comprises a light chain variable region comprising a L-CDR1 listed above, a L-CDR2 listed above and a L-CDR3 listed above, and a heavy chain variable region comprising a H-CDR1 listed above, a H-CDR2 listed above and a H-CDR3 listed above.
  • the present disclosure further provides an anti-IL-23p19 antibody or antigen-binding fragment thereof, wherein the antibody or antigen-binding fragment thereof comprises a L-CDR1, a L-CDR2, a L-CDR3, a H-CDR1, a H-CDR2 and a H-CDR3 sequence of SEQ ID NO:1, 2, 3, 33, 34, and 35, respectively; or a L-CDR1, a L-CDR2, a L-CDR3, a H-CDR1, a H-CDR2 and a H-CDR3 sequence of SEQ ID NO:4, 5, 3, 36, 34 and 37, respectively; or a L-CDR1, a L-CDR2, a L-CDR3, a H-CDR1, a H-CDR2 and a H-CDR3 sequence of SEQ ID NO:1, 2, 3, 38, 39 and 35, respectively; or a L-CDR1, a L-CDR2, a L-CDR3, a H-CDR1, a H
  • the anti-IL-23p19 antibody or antigen-binding fragment thereof comprises a light chain variable region comprising a L-CDR1, L-CDR2 and L-CDR3 combination listed above, and a heavy chain variable region comprising a H-CDR1, H-CDR2 and H-CDR3 combination listed above.
  • the present disclosure further provides an anti-IL-23p19 antibody or antigen-binding fragment thereof, wherein the antibody or antigen-binding fragment thereof comprises a light chain variable region comprising the amino acid sequence of SEQ ID NO:84 and a heavy chain variable region comprising the amino acid sequence SEQ ID NO:121 ; or a light chain variable region comprising the amino acid sequence of SEQ ID NO:86 and a heavy chain variable region comprising the amino acid sequence SEQ ID NO:123; or a light chain variable region comprising the amino acid sequence of SEQ ID NO:88 and a heavy chain variable region comprising the amino acid sequence SEQ ID NO:125; or a light chain variable region comprising the amino acid sequence of SEQ ID NO:90 and a heavy chain variable region comprising the amino acid sequence SEQ ID NO:127; or a light chain variable region comprising the amino acid sequence of SEQ ID NO:91 and a heavy chain variable region comprising the amino acid sequence SEQ ID NO:128; or a light chain variable region comprising the amino acid sequence of
  • the present disclosure further provides an anti-IL-23p19 antibody or antigen-binding fragment thereof, wherein the antibody or antigen-binding fragment thereof comprises a light chain variable region comprising an amino acid sequence selected from the group consisting of SEQ ID NO:158, 160, 162 and 164 and a heavy chain variable region comprising an amino acid sequence selected from the group consisting of SEQ ID NO:166, 168, 170 and 172.
  • the present disclosure further provides an anti-IL-23p19 antibody or antigen-binding fragment thereof, wherein the antibody or antigen-binding fragment thereof has a K D for IL-23 of less than 40pM, or a K D for IL-23 of less than 20pM, or K D for IL-23 of less than 10pM or K D for IL-23 of less than 1pM.
  • the present disclosure provides an anti-IL-23p19 antibody or antigen-binding fragment thereof that binds to human IL-23p19 at an epitope consisting of amino acid residues 108 to 126 and amino acid residues 137 to 151 of SEQ ID NO: 181.
  • present disclosure provides an anti-IL-23p19 antibody or antigen-binding fragment thereof that competitively binds to human IL-23p19 with an antibody of the present disclosure
  • present disclosure provides an anti-IL-23p19 antibody or antigen-binding fragment thereof that competitively binds to human IL-23p19 with a humanized monoclonal anti-IL-23p19 antibody comprising a light chain comprising the amino acid sequence of SEQ ID NO:174 and a heavy chain comprising the amino acid sequence of SEQ ID NO:178.
  • the present disclosure provides an anti-IL-23p19 antibody or antigen-binding fragment thereof that competitively binds to human IL-23p19 with a humanized monoclonal anti-IL-23p19 antibody comprising a light chain comprising the amino acid sequence of SEQ ID NO:180 and a heavy chain comprising the amino acid sequence of SEQ ID NO:176.
  • the present disclosure provides an anti-IL-23p19 antibody or antigen-binding fragment thereof that competitively binds to human IL-23p19 with a humanized monoclonal anti-IL-23p19 antibody comprising a light chain comprising the amino acid sequence of SEQ ID NO: 180 and a heavy chain comprising the amino acid sequence of SEQ ID NO: 178.
  • the anti-IL-23p19 antibody is a humanized antibody.
  • the anti-IL-23p19 antibody is a monoclonal antibody.
  • the anti-IL-23p19 antibody is a full length antibody.
  • the anti-IL-23p19 antibody is a humanized monoclonal antibody, for example a full length humanized monoclonal antibody.
  • the antigen-binding fragment is a Fab, F(ab') 2 , or single chain Fv fragment.
  • the antigen-binding fragment comprises a light chain variable region and a heavy chain variable region.
  • the present disclosure further provides an anti-IL-23p19 antibody or antigen-binding fragment thereof, wherein the antibody or antigen-binding fragment thereof comprises the amino acid sequence of SEQ ID NO:19 (CDR1-L); the amino acid sequence of SEQ ID NO:20 (CDR2-L) ; the amino acid sequence of SEQ ID NO:21 (CDR3-L) : the amino acid sequence of SEQ ID NO:63, 66, 67 or 68 (CDR1-H) ; the amino acid sequence of SEQ ID NO:64 (CDR2-H); and the amino acid sequence of SEQ ID NO:65 (COR3-H) .
  • the antibody or antigen-binding fragment thereof comprises the amino acid sequence of SEQ ID NO:19 (CDR1-L); the amino acid sequence of SEQ ID NO:20 (CDR2-L) ; the amino acid sequence of SEQ ID NO:21 (CDR3-L) : the amino acid sequence of SEQ ID NO:63, 66, 67 or 68 (CDR1
  • the present disclosure further provides an anti-IL-23p19 antibody or antigen-binding fragment thereof, wherein the antibody or antigen-binding fragment thereof comprises the amino acid sequence of SEQ ID NO:19 (CDR1-L); the amino acid sequence of SEQ ID NO:20 (CDR2-L); the amino acid sequence of SEQ ID NO:21 (CDR3-L) : the amino acid sequence of SEQ ID NO:66 (CDR1-H) ; the amino acid sequence of SEQ ID NO:64 (CDR2-H); and the amino acid sequence of SEQ ID NO:65 (CDR3-H) .
  • the antibody or antigen-binding fragment thereof comprises the amino acid sequence of SEQ ID NO:19 (CDR1-L); the amino acid sequence of SEQ ID NO:20 (CDR2-L); the amino acid sequence of SEQ ID NO:21 (CDR3-L) : the amino acid sequence of SEQ ID NO:66 (CDR1-H) ; the amino acid sequence of SEQ ID NO:64 (CDR2-H);
  • the present disclosure further provides an anti-IL-23p19 antibody or antigen-binding fragment thereof, wherein the antibody or antigen-binding fragment thereof comprises a light chain variable region comprising the amino acid sequence of SEQ ID NO:19 (CDR1-L); the amino acid sequence of SEQ ID NO:20 (CDR2-L); and the amino acid sequence of SEQ ID NO:21 (CDR3-L) ; and a heavy chain variable region comprising the amino acid sequence of SEQ ID NO: 63, 66, 67 or 68 (CDR1- H); the amino acid sequence of SEQ ID NO:64 (CDR2-H); and the amino acid sequence of SEQ ID NO:65 (CDR3-H).
  • the antibody or antigen-binding fragment thereof comprises a light chain variable region comprising the amino acid sequence of SEQ ID NO:19 (CDR1-L); the amino acid sequence of SEQ ID NO:20 (CDR2-L); and the amino acid sequence of SEQ ID NO:21 (CDR3-L) ; and a heavy
  • the present disclosure further provides an anti-IL-23p19 or antigen-binding fragment thereof antibody, wherein the antibody or antigen-binding fragment thereof comprises a light chain variable region comprising the amino acid sequence of SEQ ID NO:19 (CDR1-L); the amino acid sequence of SEQ ID NO:20 (CDR2-L); and the amino acid sequence of SEQ ID NO:21 (CDR3-L) ; and a heavy chain variable region comprising the amino acid sequence of SEQ ID NO:66 (CDR1-H) ; the amino acid sequence of SEQ ID NO:64 (CDR2-H); and the amino acid sequence of SEQ ID NO:65 (CDR3-H).
  • the antibody or antigen-binding fragment thereof comprises a light chain variable region comprising the amino acid sequence of SEQ ID NO:19 (CDR1-L); the amino acid sequence of SEQ ID NO:20 (CDR2-L); and the amino acid sequence of SEQ ID NO:21 (CDR3-L) ; and a heavy chain variable region comprising the amino acid
  • the present disclosure further provides an anti-IL-23p19 antibody or antigen-binding fragment thereof, wherein the antibody or antigen-binding fragment thereof comprises a light chain variable region comprising the amino acid sequence of any one of SEQ ID NO:158, 160, 162 or 164; and a heavy chain variable region comprising the amino acid sequence any one of SEQ ID NO:166, 168, 170 or 172.
  • the present disclosure further provides an anti-IL-23p19 antibody or antigen-binding fragment thereof, wherein the antibody or antigen-binding fragment thereof comprises a light chain variable region comprising the amino acid sequence of SEQ ID NO:160 and a heavy chain variable region comprising the amino acid sequence SEQ ID NO:166.
  • the present disclosure further provides an anti-IL-23p19 antibody or antigen-binding fragment thereof, wherein the antibody or antigen-binding fragment thereof comprises a light chain variable region comprising the amino acid sequence of SEQ ID NO:160 and a heavy chain variable region comprising the amino acid sequence SEQ ID NO:168.
  • the present disclosure further provides an anti-IL-23p19 antibody or antigen-binding fragment thereof, wherein the antibody or antigen-binding fragment thereof comprises a light chain variable region comprising the amino acid sequence of SEQ ID NO:158 and a heavy chain variable region comprising the amino acid sequence SEQ ID NO:166.
  • the present disclosure further provides an anti-IL-23p19 antibody or antigen-binding fragment thereof, wherein the antibody or antigen-binding fragment thereof comprises a light chain variable region comprising the amino acid sequence of SEQ ID NO:158 and a heavy chain variable region comprising the amino acid sequence SEQ ID NO:168,
  • the present disclosure further provides an antibody comprising the amino acid sequence SEQ ID NO:166 or 168 linked to a human IgG1, IgG2, IgG3, IgG4, IgM, IgA or IgE heavy chain constant region.
  • the present disclosure further provides an antibody comprising the amino acid sequence of SEQ ID NO:166 or 168 linked to a human IgG1 heavy chain constant region; and the amino acid sequence of SEQ ID NO:158 or 160 linked to a human kappa light chain constant region.
  • the present disclosure further provides a humanized monoclonal anti-IL-23p19 antibody comprising a light chain variable region comprising the amino acid sequence selected from the group consisting of any one of SEQ ID NO:158, 160, 162 and 164 and a heavy chain variable region comprising the amino acid sequence selected from the group consisting of any one of SEQ ID NO:166, 168, 170 and 172.
  • the present disclosure further provides a humanized monoclonal anti-IL-23p19 antibody comprising a light chain variable region comprising the amino acid sequence of SEQ ID NO:160 and a heavy chain variable region comprising the amino acid sequence SEQ ID NO:166.
  • the present disclosure further provides a humanized monoclonal anti-IL-23p19 antibody comprising a light chain variable region comprising the amino acid sequence of SEQ ID NO:160 and a heavy chain variable region comprising the amino acid sequence SEQ ID NO:168.
  • the present disclosure further provides a humanized monoclonal anti-IL-23p19 antibody comprising a light chain variable region comprising the amino acid sequence of SEQ ID NO:158 and a heavy chain variable region comprising the amino acid sequence SEQ ID NO:166.
  • the present disclosure further provides a humanized monoclonal anti-IL-23p19 antibody comprising a light chain variable region comprising the amino acid sequence of SEQ ID NO:158 and a heavy chain variable region comprising the amino acid sequence SEQ ID NO:168.
  • the present invention provides a pharmaceutical composition as described in the claims, comprising a a humanized monoclonal anti-IL-23p19 antibody comprising a light chain comprising the amino acid sequence of SEQ ID NO:174 and a heavy chain comprising the amino acid sequence of SEQ ID NO:176.
  • the present disclosure relates to an anti-IL-23p19 antibody or antigen-binding fragment thereof comprising a humanized light chain variable domain comprising the CDRs of SEQ ID NO:160 and framework regions having an amino acid sequence at least 90% identical to the amino acid sequence of the framework regions of the variable domain light chain amino acid sequence of SEQ ID NO:160 and a humanized heavy chain variable domain comprising the CDRs of SEQ ID NO:166 and framework regions having an amino acid sequence at least 90% identical to the amino acid sequence of the framework regions of the variable domain heavy chain amino acid sequence of SEQ ID NO:166.
  • the anti-IL-23p19 antibody is a humanized monoclonal antibody, for example a full length humanized monoclonal antibody.
  • the present disclosure relates to an anti-IL-23p19 antibody or antigen-binding fragment thereof comprising a humanized light chain variable domain comprising the CDRs of SEQ ID NO:160 and framework regions having an amino acid sequence at least 90% identical to the amino acid sequence of the framework regions of the variable domain light chain amino acid sequence of SEQ ID NO:160 and a humanized heavy chain variable domain comprising the CDRs of SEQ ID NO:168 and framework regions having an amino acid sequence at least 90% identical to the amino acid sequence of the framework regions of the variable domain heavy chain amino acid sequence of SEQ ID NO:168.
  • the anti-IL-23p19 antibody is a humanized monoclonal antibody, for example a full length humanized monoclonal antibody.
  • the present disclosure relates to an anti-IL-23p19 antibody or antigen-binding fragment thereof comprising a humanized light chain variable domain comprising the CDRs of SEQ ID NO:158 and framework regions having an amino acid sequence at least 90% identical to the amino acid sequence of the framework regions of the variable domain light chain amino acid sequence of SEQ ID NO:158 and a humanized heavy chain variable domain comprising the CDRs of SEQ ID NO:166 and framework regions having an amino acid sequence at least 90% identical to the amino acid sequence of the framework regions of the variable domain heavy chain amino acid sequence of SEQ ID NO:166.
  • the anti-IL-23p19 antibody is a humanized monoclonal antibody, for example a full length humanized monoclonal antibody.
  • the present disclosure relates to an anti-IL-23p19 antibody or antigen-binding fragment thereof comprising a humanized light chain variable domain comprising the CDRs of SEQ ID NO:158 and framework regions having an amino acid sequence at least 90% identical to the amino acid sequence of the framework regions of the variable domain light chain amino acid sequence of SEQ ID NO:158 and a humanized heavy chain variable domain comprising the CDRs of SEQ ID NO:168 and framework regions having an amino acid sequence at least 90% identical to the amino acid sequence of the framework regions of the variable domain heavy chain amino acid sequence of SEQ ID NO:168.
  • the anti-IL-23p19 antibody is a humanized monoclonal antibody, for example a full length humanized monoclonal antibody.
  • a humanized anti-IL-23p19 antibody of the present disclosure may be further characterized by a K D for human IL-23 equal or less than 1 pM. In one aspect, there is no shift in binding on-rate in 50% human serum.
  • a humanized anti-IL-23p19 antibody of the present disclosure may be further characterized in that it blocks IL-23 binding to human IL-23R/Fc in vitro.
  • a humanized anti-IL-23p19 antibody of the present disclosure mav be further characterized in that it does not bind to human IL-12.
  • a humanized anti-IL-23p19 antibody of the present disclosure mav be further characterized in that it inhibits human IL-23 induced IL-17 production in mouse splenocytes with IC 50 's equal or less than 20 pM.
  • a humanized anti-IL-23p19 antibody of the present disclosure mav be further characterized in that it inhibits human IL-23 induced STAT3 phosphorylation in human DB cells with IC 50 's equal or less than 40 pM.
  • a humanized anti-IL-23p19 antibody of the present disclosure may be further characterized in that it has no predicted activity in ADCC/CDC.
  • a humanized anti-IL-23p19 antibody of the present disclosure may be further characterized in that it has a K D equal or less than_1 pM for cynomolgus monkey IL-23.
  • a humanized anti-IL-23p19 antibody of the present disclosure may be further characterized in that it has no cross reactivity to mouse or rat IL-23.
  • a humanized anti-IL-23p19 antibody of the present disclosure may be further characterized in that it inhibits human IL-23 induced IL-17 and IL-22 production in a mouse ear at 80% or greater inhibition of both cytokines at 1 mg/kg.
  • a humanized anti-IL-23p19 antibody of the present disclosure may be further characterized by a melting temperature of 83°C as determined by differential scanning calorimetry.
  • a humanized anti-IL-23p19 antibody of the present disclosure may be further characterized by solubility equal or greater than 100 mg/ml, as measured by UV spectroscopy and monitored by turbidity.
  • a humanized anti-IL-23p19 antibody of the present disclosure may be further characterized in that it is present in at least 90% monomer form, or in at least 92% monomer form, or in at least 95% monomer form in a buffer.
  • a humanized anti-IL-23p19 antibody of the present disclosure may be further characterized in that it remains in at least 90% monomer form, or in at least 92% monomer form, or in at least 95% monomer form in a buffer for one month or for four months.
  • the anti-IL-23p19 antibody is a humanized monoclonal antibody, for example huanized monoclonal antibody.
  • DNA molecule encoding a variable light chain region above a DNA molecule encoding a variable heavy chain region above, a DNA molecule encoding a light chain region above, or a DNA molecule encoding a heavy chain region above.
  • an expression vector containing a DNA molecule above comprises a DNA molecule encoding the constant heavy chain and/or the constant light chain, respectively, linked to the DNA molecule encoding the variable heavy chain and/or the variable light chain, respectively.
  • a host cell carrying one or more expression vectors above In one reference aspects, a host is a mammalian cell.
  • Further reference aspects encompass a method for producing an antibody or antigen-binding fragment thereof above comprising transfecting a mammalian host cell with one or more of the vectors above, cultivating the host cell and recovering and purifying the antibody or antigen-binding fragment thereof.
  • Further reference aspects encompass a method for producing an antibody or antigen-binding fragment thereof above comprising obtaining a mammalian host cell comprising one or more of the vectors above, and cultivating the host cell. In one embodiment, the method further comprises recovering and purifying the antibody or antigen-binding fragment thereof.
  • the present invention provides a pharmaceutical composition as defined in the claims, for use in medicine, as defined in the claims.
  • the antibody is Antibody A, Antibody B, Antibody C or Antibody D.
  • the use is the treatment of an inflammatory disease, of an autoimmune disease, of a respiratory disease, of a metabolic disorder or of cancer.
  • the use is for the treatment of psoriasis, inflammatory bowel disease (Crohn's disease, ulcerative colitis), psoriatic arthritis, multiple sclerosis, rheumatoid arthritis, ankylosing spondylitis or Kawasaki syndrome.
  • the use is for the treatment of psoriasis.
  • the use is for the treatment of inflammatory bowel disease, for example Crohn's disease.
  • the use is for the treatment of ankylosing spondylitis.
  • the use is for the treatment of plaque psoriasis, for example chronic plaque psoriasis, for example moderate to severe chronic plaque psoriasis, for example in adult patients.
  • the patients are candidates for systemic therapy or phototherapy.
  • the use is for the treatment of moderate to severe chronic plaque psoriasis, for example in patients who are candidates for systemic therapy or phototherapy, for example adult patients.
  • the use is for the treatment of palmar pustular psoriasis, guttate psoriasis, inverse psoriasis, pustular psoriasis or erythodermic psoriasis.
  • the use is for the treatment of Crohn's disease.
  • the use is for reducing signs and symptoms, improving mucosal healing and inducing and maintaining clinical remission in adult patients with moderately to severely active Crohn's disease, in particular in patients who have had an inadequate response to conventional therapy.
  • the use is for reducing the number of draining enterocutaneous and rectovaginal fistulas and maintaining fistula closure in adult patients with fistulizing Crohn's disease.
  • the use is for the treatment of spondyloarthritis. In a further embodiment, the use is for the treatment of ankylosing spondylitis. In a further aspect, the use is for reducing signs and symptoms in patients with active ankylosing spondylitis. In a further aspect, the use is for the treatment of severe, radiographic active ankylosing spondylitis in adult patients, in particular, patients who have responded inadequately to conventional therapy.
  • the use is for the treatment of non-radiographic axial spondyloarthritis.
  • the use is for the treatment of adults with severe axial spondyloarthritis without radiographic evidence of AS but with objective signs of inflammation by elevated CRP and / or MRI, in particular patients who have had an inadequate response to, or are intolerant to nonsteroidal anti-inflammatory drugs.
  • the use is for the treatment of peripheral spondyloarthritis.
  • the use is for the treatment of discoid lupus, malaria, malignant melanoma, aplastic anaemia, Huntington's Disease, palmoplantar pustulosis, IgA nephritis, ANCA-Associated Vasculitis, scleritis or sepsis.
  • the use is for the treatment of palmoplantar pustulosis. In one reference aspect, the use is for the treatment of systemic scleroderma. In one embodiment, the antibody is Antibody A
  • the present invention further provides a pharmaceutical composition comprising an antibody molecule or antigen-binding fragment above and a pharmaceutically acceptable carrier, as described in the claims, in particular a pharmaceutical composition comprising an antibody, wherein said antibody comprises a light chain comprising the amino acid sequence of SEQ ID NO: 174 and a heavy chain comprising the amino acid sequence of SEQ ID NO: 176, wherein said pharmaceutical composition comprises:
  • the disease is plaque psoriasis, for example chronic plaque psoriasis, for example moderate to severe chronic plaque psoriasis.
  • the patients are adult patients, for example who are candidates for systemic therapy or phototherapy.
  • the disease is moderate to severe chronic plaque psoriasis.
  • the disease is palmar pustular psoriasis, guttate psoriasis, inverse psoriasis, pustular psoriasis or erythodermic psoriasis.
  • the present disclosure provides a method for reducing signs and symptoms, improving mucosal healing and inducing and maintaining clinical remission in adult patients with moderately to severely active Crohn's disease, in particular in patients who have had an inadequate response to conventional therapy, said method comprising administering to the patient an effective amount of an anti-IL-23p19 antibody or antigen-binding fragment thereof, in particular an anti-IL-23p19 antibody or antigen-binding fragment thereof above, or a pharmaceutical composition comprising the antibody or antigen-binding fragment thereof, for example a pharmaceutical composition disclosed herein.
  • the antibody is Antibody A
  • the present disclosure provides a method for reducing the number of draining enterocutaneous and rectovaginal fistulas and maintaining fistula closure in adult patients with fistulizing Crohn's disease comprising administering to the patient an effective amount of an anti-IL-23p19 antibody or antigen-binding fragment thereof above, or a pharmaceutical composition comprising the antibody or antigen-binding fragment thereof, for example a pharmaceutical composition disclosed herein.
  • the present invention provides a method for the treatment of spondyloarthritis. In one embodiment, the present invention provides a method for the treatment of ankylosing spondylitis. In a further aspect, the present disclosure provides a method for reducing signs and symptoms in patients with active ankylosing spondylitis.
  • the above methods comprise administering to a subject in need thereof, for example a patient, a pharmaceutical composition comprising the antibody as defined in the claims.
  • the antibody is Antibody A, Antibody B, Antibody C or Antibody D.
  • the present invention provides a method for the treatment of severe, radiographic active ankylosing spondylitis in adult patients, in particular, patients who have responded inadequately to conventional therapy, comprising administering to the patient a pharmaceutical composition comprising the antibody as defined in the claims .
  • the antibody is Antibody A, Antibody B, Antibody C or Antibody D.
  • the present invention provides a method for the treatment of non-radiographic axial spondyloarthritis.
  • the present invention provides a method for the treatment of adults with severe axial spondyloarthritis without radiographic evidence of AS but with objective signs of inflammation by elevated CRP and / or MRI, in particular patients who have had an inadequate response to, or are intolerant to nonsteroidal anti-inflammatory drugs.
  • the present invention provides a method for the treatment of peripheral spondyloarthritis.
  • the above methods comprise administering to a subject in need thereof, for example a a pharmaceutical composition comprising the antibody as defined in the claims.
  • the antibody is Antibody A
  • the disease is discoid lupus, malaria, malignant melanoma, aplastic anaemia, Huntington's Disease, palmoplantar pustulosis, IgA nephritis, ANCA-Associated Vasculitis, scleritis or sepsis.
  • the disease is palmoplantar pustulosis. In one embodiment, the disease is systemic scleroderma.
  • the disclosure further provides a method for inhibiting the binding of IL-23 to the IL-23 receptor on a mammalian cell, comprising administering to the cell an antibody molecule or antigen-binding fragment above, whereby signaling mediated by the IL-23 receptor is inhibited.
  • the present invention further provides a method for treating a subject having an IL-23-associated disorder, comprising administering to the subject a pharmaceutical composition comprising the anti-IL-23 antibody as described in the claims.
  • the present further provides a method for detecting and/or quantifying IL-23 levels in a biological sample by contacting the sample with an antibody or antigen binding fragment above and detecting binding of the antibody or fragment thereof with IL-23p19.
  • This information can be used to diagnose an IL-23-associated disorder.
  • methods are provided for diagnosing an IL-23-associated disorder or for determining if a subject has an increased risk of developing an IL-23-associated disorder, wherein the method comprises contacting a biological sample from a subject with an antibody or antigen binding fragment above and detecting binding of the antibody or antigen binding fragment to IL-23p19 to determine the expression or concentration of IL-23.
  • the present disclosure further provides a method for inhibiting the binding of IL-23 to the IL-23 receptor on a cell, comprising administering to the cell or cellular environment an antibody or antigen-binding fragment above, whereby signaling mediated by the IL-23 receptor is inhibited.
  • the p19 subunit of IL-23 (also referred to herein as "IL-23p19” and "p19 subunit”) is a 189 amino acid polypeptide containing a 21 aa leader sequence ( Oppmann et al. Immunity 13:715 (2000 ), SEQ ID NO: 181). The biological activity of the molecule is only detected when it is partnered with the IL-12p40 subunit to form IL-23. IL-23 is predominantly expressed by activated dendritic cells (DCs) and phagocytic cells.
  • the receptor for IL-23 was found to be composed of the IL-12R ⁇ 1 subunit of IL-12 receptor partnered with a unique subunit called IL-23R ( Parham et al. J.
  • IL-17A IL-17A
  • IL-17F IL-22
  • IL-22 pro-inflammatory cytokines
  • IL-6 pro-inflammatory cytokines
  • TNF- ⁇ pro-inflammatory cytokines
  • the present disclosure provides antibodies that bind to the p19 subunit of IL-23, in particular IL-23p19. Also disclosed herein are humanized antibodies that recognize the p19 subunit of IL-23. In specific reference aspects, the sequence of these humanized antibodies has been identified based on the sequences of certain lead mouse antibodies.
  • the lead mouse antibodies of the present disclosure were derived from mouse hybridomas.
  • the immunization of the mice is carried out using different techniques.
  • antibodies that are specific for human IL-23p19 proteins or fragments thereof can be raised against an immunogenic antigen such as an isolated IL-23p19 protein, an isolated IL-23 protein, an isolated hybrid IL-23 protein, and/or a portion thereof of any of the above (including synthetic peptides).
  • an immunogenic antigen such as an isolated IL-23p19 protein, an isolated IL-23 protein, an isolated hybrid IL-23 protein, and/or a portion thereof of any of the above (including synthetic peptides).
  • a hybrid IL-23 protein comprising a mouse IL-23p40 subunit and a human IL-23p19 subunit is used to immunize mice.
  • Preparation of immunogenic antigens and monoclonal antibody production can be performed using any suitable technique known in the art.
  • the present invention provides an antibody that binds to human IL-23 with high affinity. Selected mouse antibodies were humanized to result in humanized antibodies.
  • the humanized antibodies of the present disclosure bind to human IL-23 with high affinity.
  • the present disclosure provides a humanized antibody that binds to human IL-23 with high affinity.
  • the present disclosure provides an anti-IL-23p19 antibody having a K D of less than 40pM.
  • the present disclosure provides an anti-IL-23p19 antibody having a K D of less than 20pM.
  • the present disclosure provides an anti-IL-23p19 antibody having a K D less than 10pM.
  • the present disclosure provides an anti-IL-23p19 antibody having a K D less than 1pM.
  • an antibody of the present disclosure binds to IL-23p19 with high affinity in the absence of human serum or in the presence of 50% human serum.
  • a humanized antibody of the present disclosure also binds to cynomolgus monkey IL-23 with high affinity.
  • an antibody of the present disclosure binds to IL-23, but does not bind to IL-12. In a further aspect, an antibody of the present disclosure does not interfere with the biological activity of IL-12, which is a closely related family member to IL-23.
  • an antibody of the present disclosure inhibits the IL-23 stimulated production of IL-17 from mouse splenocytes.
  • a humanized antibody of the present disclosure inhibits IL-23-induced STAT3 phosphorylation in DB cells.
  • a humanized antibody of the present disclosure antagonizes the action of IL-23 by binding to the p19 subunit of IL-23, as measured by the inhibition of cytokines such as IL-17 and IL-22, whose production is stimulated by IL-23, and detected by the reduction in the levels of these cytokine
  • a humanized antibody of the present disclosure has a favorable pharmacokinetic profile (PK) profile, as exemplified by in vivo half life in cynomolgus monkeys.
  • PK pharmacokinetic profile
  • a humanized monoclonal anti-IL-23p19 antibody of the present disclosure has favorable biophysical properties, for example quality, stability, or solubility.
  • the anti-IL-23p19 antibody is a humanized antibody.
  • the anti-IL-23p19 antibody is a monoclonal antibody.
  • the anti-IL-23p19 antibody is a full length antibody.
  • the anti-IL-23p19 antibody is a humanized monoclonal antibody, for example a full length humanized monoclonal antibody.
  • an antibody or antigen-binding fragment thereof of the present disclosure recognizes specific "IL-23p19 antigen epitope" or " IL-23p19 epitope”.
  • these terms refer to a molecule (e.g., a peptide) or a fragment of a molecule capable of immunoreactivity with an anti-IL-23p19 antibody and, for example, include an IL-23p19 antigenic determinant recognized by the any of the antibodies having a light chain/heavy chain sequence combination of SEQ ID NO:84/121, 86/123, 88/125, 90/127, 91/128, 93/130, 95/132, 97/134, 99/136, 101/138, 103/140, 105/142, 107/144, 109/146, 111/148, 113/150, 115/152, 117/154, 119/156, 160/166, 160/168, 158/166 or 158/168.
  • IL-23p19 antigen epitopes can be included in proteins, protein fragments, peptides or the like.
  • the epitopes are most commonly proteins, short oligopeptides, oligopeptide mimics (i.e., organic compounds that mimic antibody binding properties of the IL-23p19 antigen), or combinations thereof.
  • the minimum size of a peptide or polypeptide epitope for an antibody is thought to be about four to five amino acids.
  • Peptide or polypeptide epitopes contain for example at least seven amino acids or for example at least nine amino acids or for example between about 15 to about 20 amino acids.
  • Epitopes may be determined by various techniques known in the art, such as X-ray crystallography, Hydrogen/Deuterium Exchange Mass Spectrometry (HXMS), site-directed mutagenesis, alanine scanning mutagenesis, and peptide screening methods.
  • HXMS Hydrogen/Deuterium Exchange Mass Spectrometry
  • antibodies or immunoglobulin are heterotetrameric glycoproteins, typically of about 150,000 daltons, composed of two identical light (L) chains and two identical heavy (H) chains and are typically referred to as full length antibodies.
  • Each light chain is covalently linked to a heavy chain by one disulfide bond to form a heterodimer, and the heterotrameric molecule is formed through a covalent disulfide linkage between the two identical heavy chains of the heterodimers.
  • the light and heavy chains are linked together by one disulfide bond, the number of disulfide linkages between the two heavy chains varies by immunoglobulin isotype.
  • Each heavy and light chain also has regularly spaced intrachain disulfide bridges.
  • Each heavy chain has at the amino-terminus a variable domain (V H ), followed by three or four constant domains (C H1 , C H2 , C H3 , and C H4 ), as well as a hinge region between C H1 and C H2 .
  • Each light chain has two domains, an amino-terminal variable domain (V L ) and a carboxy-terminal constant domain (C L ).
  • the V L domain associates non-covalently with the V H domain, whereas the C L domain is commonly covalently linked to the C H1 domain via a disulfide bond.
  • Particular amino acid residues are believed to form an interface between the light and heavy chain variable domains ( Chothia et al., 1985, J. Mol. Biol. 186:651-663 ).
  • Variable domains are also referred herein as variable regions.
  • CDRs complementarity determining regions
  • HVLs hypervariable loops
  • HVLs also referred herein as CDRs
  • CDRs are structurally defined according to the three-dimensional structure of the variable domain, as described by Chothia and Lesk, 1987, J. Mol. Biol. 196: 901-917 .
  • CDR-L1 is positioned at about residues 24-34, CDR-L2, at about residues 50-56, and CDR-L3, at about residues 89-97 in the light chain variable domain;
  • CDR-H1 is positioned at about residues 31-35, CDR-H2 at about residues 50-65, and CDR-H3 at about residues 95-102 in the heavy chain variable domain.
  • residue numbers that encompass a particular CDR will vary depending on the sequence and size of the CDR. Those skilled in the art can routinely determine which residues comprise a particular CDR given the variable region amino acid sequence of the antibody.
  • the CDR1, CDR2, CDR3 of the heavy and light chains therefore define the unique and functional properties specific for a given antibody.
  • the three CDRs within each of the heavy and light chains are separated by framework regions (FR), which contain sequences that tend to be less variable. From the amino terminus to the carboxy terminus of the heavy and light chain variable domains, the FRs and CDRs are arranged in the order: FR1, CDR1, FR2, CDR2, FR3, CDR3, and FR4.
  • FR1, CDR1, FR2, CDR2, FR3, CDR3, and FR4 The largely ⁇ -sheet configuration of the FRs brings the CDRs within each of the chains into close proximity to each other as well as to the CDRs from the other chain.
  • the resulting conformation contributes to the antigen binding site (see Kabat et al., 1991, NIH Publ. No. 91-3242, Vol. I, pages 647-669), although not all CDR residues are necessarily directly involved in antigen binding.
  • FR residues and Ig constant domains are not directly involved in antigen binding, but contribute to antigen binding and/or mediate antibody effector function. Some FR residues are thought to have a significant effect on antigen binding in at least three ways: by noncovalently binding directly to an epitope, by interacting with one or more CDR residues, and by affecting the interface between the heavy and light chains.
  • the constant domains are not directly involved in antigen binding but mediate various Ig effector functions, such as participation of the antibody in antibody dependent cellular cytotoxicity (ADCC), complement dependent cytotoxicity (CDC) and antibody dependent cellular phagocytosis (ADCP).
  • the light chains of vertebrate immunoglobulins are assigned to one of two clearly distinct classes, kappa ( ⁇ ) and lambda ( ⁇ ), based on the amino acid sequence of the constant domain.
  • the heavy chains of mammalian immunoglobulins are assigned to one of five major classes, according to the sequence of the constant domains: IgA, IgD, IgE, IgG, and IgM.
  • IgG and IgA are further divided into subclasses (isotypes), e.g., IgG 1 , IgG 2 , IgG 3 , IgG 4 , IgA 1 , and IgA 2 .
  • the heavy chain constant domains that correspond to the different classes of immunoglobulins are called ⁇ , ⁇ , ⁇ , ⁇ , and ⁇ , respectively.
  • the subunit structures and three-dimensional configurations of the classes of native immunoglobulins are well known.
  • antibody specifically encompass monoclonal antibodies (including full length monoclonal antibodies), polyclonal antibodies, multispecific antibodies (e.g., bispecific antibodies), and antibody fragments such as variable domains and other portions of antibodies that exhibit a desired biological activity, e.g., IL-23p19 binding.
  • monoclonal antibody refers to an antibody that is highly specific, being directed against a single antigenic determinant, an "epitope”.
  • monoclonal is indicative of antibodies directed to the identical epitope and is not to be construed as requiring production of the antibody by any particular method. It should be understood that monoclonal antibodies can be made by any technique or methodology known in the art; including e.g., the hybridoma method ( Kohler et al., 1975, Nature 256:495 ), or recombinant DNA methods known in the art (see, e.g., U.S. Pat. No.
  • monomer refers to a homogenous form of an antibody.
  • monomer means a monomeric antibody having two identical heavy chains and two identical light chains.
  • Chimeric antibodies consist of the heavy and light chain variable regions of an antibody from one species (e.g., a non-human mammal such as a mouse) and the heavy and light chain constant regions of another species (e.g., human) antibody and can be obtained by linking the DNA sequences encoding the variable regions of the antibody from the first species (e.g., mouse) to the DNA sequences for the constant regions of the antibody from the second (e.g. human) species and transforming a host with an expression vector containing the linked sequences to allow it to produce a chimeric antibody.
  • a non-human mammal such as a mouse
  • human constant regions of another species
  • the chimeric antibody also could be one in which one or more regions or domains of the heavy and/or light chain is identical with, homologous to, or a variant of the corresponding sequence in a monoclonal antibody from another immunoglobulin class or isotype, or from a consensus or germline sequence.
  • Chimeric antibodies can include fragments of such antibodies, provided that the antibody fragment exhibits the desired biological activity of its parent antibody, for example binding to the same epitope (see, e.g., U.S. Pat. No. 4,816,567 ; and Morrison et al., 1984, Proc. Natl. Acad. Sci. USA 81: 6851-6855 ).
  • antibody fragment refers to a portion of a full length anti-IL-23p19 antibody, in which a variable region or a functional capability is retained, for example, specific IL-23p19 epitope binding.
  • antibody fragments include, but are not limited to, a Fab, Fab', F(ab') 2 , Fd, Fv, scFv and scFv-Fc fragment, a diabody, a linear antibody, a single-chain antibody, a minibody, a diabody formed from antibody fragments, and multispecific antibodies formed from antibody fragments.
  • Full length antibodies can be treated with enzymes such as papain or pepsin to generate useful antibody fragments.
  • Papain digestion is used to produces two identical antigen-binding antibody fragments called "Fab” fragments, each with a single antigen-binding site, and a residual "Fc” fragment.
  • the Fab fragment also contains the constant domain of the light chain and the C H1 domain of the heavy chain.
  • Pepsin treatment yields a F(ab') 2 fragment that has two antigen-binding sites and is still capable of cross-linking antigen.
  • Fab' fragments differ from Fab fragments by the presence of additional residues including one or more cysteines from the antibody hinge region at the C-terminus of the C H1 domain.
  • F(ab') 2 antibody fragments are pairs of Fab' fragments linked by cysteine residues in the hinge region. Other chemical couplings of antibody fragments are also known.
  • Fv fragment contains a complete antigen-recognition and binding site consisting of a dimer of one heavy and one light chain variable domain in tight, non-covalent association.
  • the three CDRs of each variable domain interact to define an antigen-biding site on the surface of the V H -V L dimer.
  • the six CDRs confer antigen-binding specificity to the antibody.
  • a “single-chain Fv” or “scFv” antibody fragment is a single chain Fv variant comprising the V H and V L domains of an antibody where the domains are present in a single polypeptide chain.
  • the single chain Fv is capable of recognizing and binding antigen.
  • the scFv polypeptide may optionally also contain a polypeptide linker positioned between the V H and V L domains in order to facilitate formation of a desired three-dimensional structure for antigen binding by the scFv (see, e.g., Pluckthun, 1994, In The Pharmacology of monoclonal Antibodies, Vol. 113, Rosenburg and Moore eds., Springer-Verlag, New York, pp. 269-315 ).
  • a “diabody” refers to small antibody fragments with two antigen-binding sites, which fragments comprise a heavy chain variable domain (V.sub.H) connected to a light chain variable domain (V.sub.L) in the same polypeptide chain (V.sub.H-V.sub.L or V.sub.L-V.sub.H).
  • V.sub.H heavy chain variable domain
  • V.sub.L light chain variable domain
  • Diabodies are described more fully in, e.g., Holliger et al. (1993) Proc. Natl. Acad. Sci. USA 90: 6444-6448 .
  • Other recognized antibody fragments include those that comprise a pair of tandem Fd segments (V H -C H1 -V H -C H1 ) to form a pair of antigen binding regions.
  • These "linear antibodies” can be bispecific or monospecific as described in, for example, Zapata et al. 1995, Protein Eng. 8(10):1057-1062 .
  • a “humanized antibody” or a “humanized antibody fragment” is a specific type of chimeric antibody which includes an immunoglobulin amino acid sequence variant, or fragment thereof, which is capable of binding to a predetermined antigen and which, comprises one or more FRs having substantially the amino acid sequence of a human immunoglobulin and one or more CDRs having substantially the amino acid sequence of a non-human immunoglobulin.
  • This non-human amino acid sequence often referred to as an "import” sequence is typically taken from an "import" antibody domain, particularly a variable domain.
  • a humanized antibody includes at least the CDRs or HVLs of a non-human antibody, inserted between the FRs of a human heavy or light chain variable domain.
  • the present disclosure describes specific humanized anti-IL-23p19 antibodies which contain CDRs derived from the mouse monoclonal antibodies or humanized CDRs shown in Tables 3 and 4 inserted between the FRs of human germline sequence heavy and light chain variable domains. It will be understood that certain mouse FR residues may be important to the function of the humanized antibodies and therefore certain of the human germline sequence heavy and light chain variable domains residues are modified to be the same as those of the corresponding mouse sequence.
  • a humanized anti-IL-23p19 antibody comprises substantially all of at least one, and typically two, variable domains (such as contained, for example, in Fab, Fab', F(ab')2, Fabc, and Fv fragments) in which all, or substantially all, of the CDRs correspond to those of a non-human immunoglobulin, and specifically herein, all of the CDRs are mouse or humanized sequences as detailed in Tables 1 through 4 herein below and all, or substantially all, of the FRs are those of a human immunoglobulin consensus or germline sequence.
  • a humanized anti- IL-23p19 antibody also includes at least a portion of an immunoglobulin Fc region, typically that of a human immunoglobulin.
  • the antibody will contain both the light chain as well as at least the variable domain of a heavy chain.
  • the antibody also may include one or more of the C H1 , hinge, C H2 , C H3 , and/or C H4 regions of the heavy chain, as appropriate.
  • a humanized anti-IL-23p19 antibody can be selected from any class of immunoglobulins, including IgM, IgG, IgD, IgA and IgE, and any isotype, including IgG 1 , IgG 2 , IgG 3 , IgG 4 , IgA 1 and IgA 2 .
  • the constant domain can be a complement fixing constant domain where it is desired that the humanized antibody exhibit cytotoxic activity, and the isotype is typically IgG 1 . Where such cytotoxic activity is not desirable, the constant domain may be of another isotype, e.g., IgG 2 .
  • An alternative humanized anti-IL-23p19 antibody can comprise sequences from more than one immunoglobulin class or isotype, and selecting particular constant domains to optimize desired effector functions is within the ordinary skill in the art.
  • the present disclosure provides antibodies that are IgG1 antibodies and more particularly, are IgG1 antibodies in which there is a knock-out of effector functions.
  • the FRs and CDRs, or HVLs, of a humanized anti-IL-23p19 antibody need not correspond precisely to the parental sequences.
  • one or more residues in the import CDR, or HVL, or the consensus or germline FR sequence may be altered (e.g., mutagenized) by substitution, insertion or deletion such that the resulting amino acid residue is no longer identical to the original residue in the corresponding position in either parental sequence but the antibody nevertheless retains the function of binding to IL-23p19.
  • Such alteration typically will not be extensive and will be conservative alterations.
  • at least 75% of the humanized antibody residues will correspond to those of the parental consensus or germline FR and import CDR sequences, more often at least 90%, and most frequently greater than 95%, or greater than 98% or greater than 99%.
  • V L -V H interface Immunoglobulin residues that affect the interface between heavy and light chain variable regions
  • Certain residues that may be involved in interchain interactions include V L residues 34, 36, 38, 44, 46, 87, 89, 91, 96, and 98 and V H residues 35, 37, 39, 45, 47, 91, 93, 95, 100, and 103 (utilizing the numbering system set forth in Kabat et al., Sequences of Proteins of Immunological Interest (National Institutes of Health, Bethesda, Md., 1987 )).
  • V L residues 34, 36, 38, 44, 46, 87, 89, 91, 96, and 98 V H residues 35, 37, 39, 45, 47, 91, 93, 95, 100, and 103
  • V L residues 43 and 85 residues such as V L residues 43 and 85, and V H residues 43 and 60 also may be involved in this interaction. While these residues are indicated for human IgG only, they are applicable across species. Important antibody residues that are reasonably expected to be involved in interchain interactions are selected for substitution into the consensus sequence.
  • Consensus sequence and “consensus antibody” refer to an amino acid sequence which comprises the most frequently occurring amino acid residue at each location in all immunoglobulins of any particular class, isotype, or subunit structure, e.g., a human immunoglobulin variable domain.
  • the consensus sequence may be based on immunoglobulins of a particular species or of many species.
  • a "consensus” sequence, structure, or antibody is understood to encompass a consensus human sequence as described in certain embodiments, and to refer to an amino acid sequence which comprises the most frequently occurring amino acid residues at each location in all human immunoglobulins of any particular class, isotype, or subunit structure.
  • the consensus sequence contains an amino acid sequence having at each position an amino acid that is present in one or more known immunoglobulins, but which may not exactly duplicate the entire amino acid sequence of any single immunoglobulin.
  • the variable region consensus sequence is not obtained from any naturally produced antibody or immunoglobulin. Kabat et al., 1991, Sequences of Proteins of Immunological Interest, 5th Ed. Public Health Service, National Institutes of Health, Bethesda, Md., and variants thereof.
  • the FRs of heavy and light chain consensus sequences, and variants thereof provide useful sequences for the preparation of humanized anti-IL-23p19 antibodies. See, for example, U.S. Pat. Nos. 6,037,454 and 6,054,297 .
  • Germline antibody sequences for the light chain of the antibody come from conserved human germline kappa or lambda v-genes and j-genes.
  • the heavy chain sequences come from germline v-, d- and j-genes ( LeFranc, M-P, and LeFranc, G, "The Immunoglobulin Facts Book” Academic Press, 2001 ).
  • variant each refers to a humanized anti-IL-23p19 antibody having at least a light chain variable murine CDR from any of the sequences as shown in Table 1 or a heavy chain murine CDR sequence derived from the murine monoclonal antibody as shown in Table 2.
  • Variants include those having one or more amino acid changes in one or both light chain or heavy chain variable domains, provided that the amino acid change does not substantially impair binding of the antibody to IL-23p19.
  • Exemplary humanized antibodies produced herein include those designated as Antibody A, Antibody B, Antibody C and Antibody D, and the various light chains and heavy chains of the same are shown in SEQ ID Nos:174 and 180, and SEQ ID Nos:176 and 178, respectively.
  • an “isolated” antibody is one that has been identified and separated and/or recovered from a component of its natural environment. Contaminant components of the antibody's natural environment are those materials that may interfere with diagnostic or therapeutic uses of the antibody, and can be enzymes, hormones, or other proteinaceous or nonproteinaceous solutes. In one aspect, the antibody will be purified to at least greater than 95% isolation by weight of antibody.
  • An isolated antibody includes an antibody in situ within recombinant cells in which it is produced, since at least one component of the antibody's natural environment will not be present. Ordinarily however, an isolated antibody will be prepared by at least one purification step in which the recombinant cellular material is removed.
  • antibody performance refers to factors that contribute to antibody recognition of antigen or the effectiveness of an antibody in vivo. Changes in the amino acid sequence of an antibody can affect antibody properties such as folding, and can influence physical factors such as initial rate of antibody binding to antigen (k a ), dissociation constant of the antibody from antigen (k d ), affinity constant of the antibody for the antigen (Kd), conformation of the antibody, protein stability, and half life of the antibody.
  • epitope tagged when used herein, refers to an anti-IL-23p19 antibody fused to an "epitope tag".
  • An “epitope tag” is a polypeptide having a sufficient number of amino acids to provide an epitope for antibody production, yet is designed such that it does not interfere with the desired activity of the humanized anti-IL-23p19 antibody.
  • the epitope tag is usually sufficiently unique such that an antibody raised against the epitope tag does not substantially cross-react with other epitopes.
  • Suitable tag polypeptides generally contain at least 6 amino acid residues and usually contain about 8 to 50 amino acid residues, or about 9 to 30 residues.
  • epitope tags and the antibody that binds the epitope include the flu HA tag polypeptide and its antibody 12CA5 ( Field et al., 1988 Mol. Cell. Biol. 8: 2159-2165 ; c-myc tag and 8F9, 3C7, 6E10, G4, B7 and 9E10 antibodies thereto ( Evan et al., 1985, Mol. Cell. Biol. 5(12):3610-3616 ; and Herpes simplex virus glycoprotein D (gD) tag and its antibody ( Paborsky et al. 1990, Protein Engineering 3(6): 547-553 ).
  • the epitope tag is a "salvage receptor binding epitope".
  • the term "salvage receptor binding epitope” refers to an epitope of the Fc region of an IgG molecule (such as IgG 1 , IgG 2 , IgG 3 , or IgG 4 ) that is responsible for increasing the in vivo serum half-life of the IgG molecule.
  • the antibodies of the present disclosure may be conjugated to a cytotoxic agent.
  • a cytotoxic agent This is any substance that inhibits or prevents the function of cells and/or causes destruction of cells.
  • the term is intended to include radioactive isotopes (such as I 131 , I 125 , Y 90 , and Re 186 ), chemotherapeutic agents, and toxins such as enzymatically active toxins of bacterial, fungal, plant, or animal origin, and fragments thereof.
  • cytotoxic agents can be coupled to the humanized antibodies of the present disclosure using standard procedures, and used, for example, to treat a patient indicated for therapy with the antibody.
  • chemotherapeutic agent is a chemical compound useful in the treatment of cancer.
  • chemotherapeutic agents include alkylating agents such a thiotepa and cyclosphosphamide; alkyl sulfonates such as busulfan, improsulfan, and piposulfan; aziridines such as benzodopa, carboquone, meturedopa, and uredopa; ethylenimines and methylamelamines including altretamine, triethylenemelamine, trietylenephosphoramide, triethylenethiophosphoramide, and trimethylolomelamine; acetogenins (especially bullatacin and bullatacinone); camptothecin (including the synthetic analogue topotecan); bryostatin; callystatin; CC-1065 (including its adozelesin, carze
  • dynemicin including dynemicin A; bisphosphonates, such as clodronate; esperamicin; as well as neocarzinostatin chromophore and related chromoprotein enediyne antibiotic chromomophores), aclacinomysins, actinomycin, authramycin, azaserine, bleomycins, cactinomycin, carabicin, caminomycin, carzinophilin, chromomycins, dactinomycin, daunorubicin, detorubicin, 6-diazo-5-oxo-L-norleucine, doxorubicin (Adriamycin TM ) (including morpholino-doxorubicin, cyanomorpholino-doxorubicin, 2-pyrrolino-doxorubicin, and deoxydoxorubicin),
  • anti-hormonal agents that act to regulate or inhibit hormone action on tumors
  • SERMs selective estrogen receptor modulators
  • tamoxifen including Nolvadex TM
  • raloxifene including Nolvadex TM
  • droloxifene 4-hydroxytamoxifen
  • trioxifene keoxifene
  • LY117018 onapristone
  • toremifene Fraston TM
  • aromatase inhibitors that inhibit the enzyme aromatase, which regulates estrogen production in the adrenal glands, such as, for example, 4(5)-imidazoles, aminoglutethimide, megestrol acetate (Megace TM ), exemestane, formestane, fadrozole, vorozole (Rivisor TM ), letrozole (Femara TM ), and anastrozole (Arimidex TM ); and anti-androgens such as flu
  • the antibodies also may be conjugated to prodrugs.
  • a "prodrug” is a precursor or derivative form of a pharmaceutically active substance that is less cytotoxic to tumor cells compared to the parent drug and is capable of being enzymatically activated or converted into the more active form. See, for example, Wilman, 1986, “Prodrugs in Cancer Chemotherapy", In Biochemical Society Transactions, 14, pp. 375-382 , 615th Meeting Harbor and Stella et al., 1985, “Prodrugs: A Chemical Approach to Targeted Drug Delivery, In: “Directed Drug Delivery, Borchardt et al., (ed.), pp. 247-267, Humana Press .
  • Useful prodrugs include, but are not limited to, phosphate-containing prodrugs, thiophosphate-containing prodrugs, sulfate-containing prodrugs peptide-containing prodrugs, D-amino acid-modified prodrugs, glycosylated prodrugs, ⁇ -lactam-containing prodrugs, optionally substituted phenoxyacetamide-containing prodrugs, and optionally substituted phenylacetamide-containing prodrugs, 5-fluorocytosine and other 5-fluorouridine prodrugs that can be converted into the more active cytotoxic free drug.
  • cytotoxic drugs that can be derivatized into a prodrug form include, but are not limited to, those chemotherapeutic agents described above.
  • the antibodies of the disclosure also may be conjugated to a label, either a label alone or a label and an additional second agent (prodrug, chemotherapeutic agent and the like).
  • a label as distinguished from the other second agents refers to an agent that is a detectable compound or composition and it may be conjugated directly or indirectly to a humanized antibody of the present disclosure.
  • the label may itself be detectable (e.g., radioisotope labels or fluorescent labels) or, in the case of an enzymatic label, may catalyze chemical alteration of a substrate compound or composition that is detectable.
  • Labeled humanized anti-IL-23p19 antibody can be prepared and used in various applications including in vitro and in vivo diagnostics.
  • the antibodies of the present disclosure may be formulated as part of a liposomal preparation in order to affect delivery thereof in vivo.
  • a "liposome” is a small vesicle composed of various types of lipids, phospholipids, and/or surfactant. Liposomes are useful for delivery to a mammal of a compound or formulation, such as a humanized anti-IL-23p19 antibody disclosed herein, optionally, coupled to or in combination with one or more pharmaceutically active agents and/or labels.
  • the components of the liposome are commonly arranged in a bilayer formation, similar to the lipid arrangement of biological membranes.
  • isolated nucleic acid molecule is a nucleic acid molecule that is identified and separated from at least one contaminant nucleic acid molecule with which it is ordinarily associated in the natural source of the antibody nucleic acid.
  • isolated nucleic acid molecule is distinguished from the nucleic acid molecule as it exists in natural cells.
  • one or more domains of the humanized antibodies will be recombinantly expressed.
  • Such recombinant expression may employ one or more control sequences, i.e., polynucleotide sequences necessary for expression of an operably linked coding sequence in a particular host organism.
  • the control sequences suitable for use in prokaryotic cells include, for example, promoter, operator, and ribosome binding site sequences.
  • Eukaryotic control sequences include, but are not limited to, promoters, polyadenylation signals, and enhancers. These control sequences can be utilized for expression and production of humanized anti-IL-23p19 antibody in prokaryotic and eukaryotic host cells.
  • a nucleic acid sequence is "operably linked" when it is placed into a functional relationship with another nucleic acid sequence.
  • a nucleic acid presequence or secretory leader is operably linked to a nucleic acid encoding a polypeptide if it is expressed as a preprotein that participates in the secretion of the polypeptide;
  • a promoter or enhancer is operably linked to a coding sequence if it affects the transcription of the sequence; or a ribosome binding site is operably linked to a coding sequence if it is positioned so as to facilitate translation.
  • "operably linked” means that the DNA sequences being linked are contiguous, and, in the case of a secretory leader, contiguous and in reading frame. However, enhancers are optionally contiguous. Linking can be accomplished by ligation at convenient restriction sites. If such sites do not exist, synthetic oligonucleotide adaptors or linkers can be used.
  • cell As used herein, the expressions "cell”, “cell line”, and “cell culture” are used interchangeably and all such designations include the progeny thereof. Thus, “transformants” and “transformed cells” include the primary subject cell and cultures derived therefrom without regard for the number of transfers.
  • mammal for purposes of treatment refers to any animal classified as a mammal, including humans, domesticated and farm animals, and zoo, sports, or pet animals, such as dogs, horses, cats, cows, and the like.
  • the mammal is human.
  • a “disorder”, as used herein, is any condition that would benefit from treatment with a humanized anti-IL-23p19 antibody described herein. This includes chronic and acute disorders or diseases including those pathological conditions that predispose the mammal to the disorder in question.
  • disorders to be treated herein include inflammatory, angiogenic, autoimmune and immunologic disorders, respiratory disorders, cancer, hematological malignancies, benign and malignant tumors, leukemias and lymphoid malignancies.
  • cancer and “cancerous” refer to or describe the physiological condition in mammals that is typically characterized by unregulated cell growth.
  • Examples of cancer include, but are not limited to, carcinoma, lymphoma, blastoma, sarcoma, and leukemia.
  • IL-23-associated disorder or "IL-23-associated disease” refers to a condition in which IL-23 activity contributes to the disease and typically where IL-23 is abnormally expressed.
  • An IL-23-associated disorder includes diseases and disorders of the immune system, such as autoimmune disorders and inflammatory disorders.
  • Such conditions include, but are not limited to, rheumatoid arthritis (RA), systemic lupus erythematosus (SLE), scleroderma, Sjogren's syndrome, multiple sclerosis, psoriasis, psoriatic arthritis, inflammatory bowel disease (e.g., ulcerative colitis and Crohn's disease), pulmonary inflammation, asthma, idiopathic thrombocytopenic purara (ITP) and spondyloarthritis, for example ankylosing spondylitis, non-radiographic axial spondyloarthritis or peripheral spondyloarthritis.
  • intravenous infusion refers to introduction of an agent into the vein of an animal or human patient over a period of time greater than approximately 15 minutes, generally between approximately 30 to 90 minutes.
  • intravenous bolus or “intravenous push” refers to drug administration into a vein of an animal or human such that the body receives the drug in approximately 15 minutes or less, generally 5 minutes or less.
  • subcutaneous administration refers to introduction of an agent under the skin of an animal or human patient, preferable within a pocket between the skin and underlying tissue, by relatively slow, sustained delivery from a drug receptacle. Pinching or drawing the skin up and away from underlying tissue may create the pocket.
  • subcutaneous infusion refers to introduction of a drug under the skin of an animal or human patient, preferably within a pocket between the skin and underlying tissue, by relatively slow, sustained delivery from a drug receptacle for a period of time including, but not limited to, 30 minutes or less, or 90 minutes or less.
  • the infusion may be made by subcutaneous implantation of a drug delivery pump implanted under the skin of the animal or human patient, wherein the pump delivers a predetermined amount of drug for a predetermined period of time, such as 30 minutes, 90 minutes, or a time period spanning the length of the treatment regimen.
  • subcutaneous bolus refers to drug administration beneath the skin of an animal or human patient, where bolus drug delivery is less than approximately 15 minutes; in another aspect, less than 5 minutes, and in still another aspect, less than 60 seconds. In yet even another aspect, administration is within a pocket between the skin and underlying tissue, where the pocket may be created by pinching or drawing the skin up and away from underlying tissue.
  • therapeutically effective amount is used to refer to an amount of an active agent that relieves or ameliorates one or more of the symptoms of the disorder being treated.
  • therapeutically effective amount refers to a target serum concentration that has been shown to be effective in, for example, slowing disease progression. Efficacy can be measured in conventional ways, depending on the condition to be treated.
  • treatment and “therapy” and the like, as used herein, are meant to include therapeutic as well as prophylactic, or suppressive measures for a disease or disorder leading to any clinically desirable or beneficial effect, including but not limited to alleviation or relief of one or more symptoms, regression, slowing or cessation of progression of the disease or disorder.
  • treatment includes the administration of an agent prior to or following the onset of a symptom of a disease or disorder thereby preventing or removing one or more signs of the disease or disorder.
  • the term includes the administration of an agent after clinical manifestation of the disease to combat the symptoms of the disease.
  • administration of an agent after onset and after clinical symptoms have developed where administration affects clinical parameters of the disease or disorder, such as the degree of tissue injury or the amount or extent of metastasis, whether or not the treatment leads to amelioration of the disease, comprises “treatment” or "therapy” as used herein.
  • treatment or “therapy” as used herein.
  • compositions of the invention either alone or in combination with another therapeutic agent alleviate or ameliorate at least one symptom of a disorder being treated as compared to that symptom in the absence of use of the humanized anti-IL-23p19 antibody composition, the result should be considered an effective treatment of the underlying disorder regardless of whether all the symptoms of the disorder are alleviated or not.
  • package insert is used to refer to instructions customarily included in commercial packages of therapeutic products, that contain information about the indications, usage, administration, contraindications and/or warnings concerning the use of such therapeutic products.
  • anti-IL-23 antibodies in particular humanized anti-IL-23p19 antibodies, and compositions and articles of manufacture comprising one or more anti-IL-23 antibody, in particular one or more humanized anti-IL-23p19 antibody of the present invention.
  • binding agents that include an antigen-binding fragment of an anti-IL-23 antibody, in particular a humanized anti-IL-23p19 antibody.
  • the humanized anti-IL-23p19 antibodies and binding agents can inhibit the production of Th17 associated cytokines, which contribute to chronic autoimmune and inflammatory diseases.
  • the humanized anti-IL-23p19 antibodies and binding agents can thus be used in the treatment of a variety of diseases or disorders.
  • a humanized anti-IL-23p19 antibody and an IL-23p19 binding agent each includes at least a portion that specifically recognizes an IL-23p19 epitope (i.e., an antigen-binding fragment).
  • mice antibodies were selected based on IL-23p19 binding characterization.
  • an antibody of the present disclosure has a K D for IL-23, in particular human IL-23, of less than 100pM.
  • an antibody of the present disclosure has a Ko of less than 40pM.
  • an antibody of the present disclosure has a K D of less than 20pM.
  • an antibody of the present disclosure has a K D of less than 10pM.
  • a monoclonal antibody of the present disclosure has a K D of less than 1pM.
  • mice antibodies have the following light chain variable regions and heavy chain variable regions as shown in Table 1 and 2: Table 1: Anti-IL-23p19 Mouse Leads - VK Sequences 2D1vk 6B8Vk 9D12-Vk 15Cllvk 15F1vk 18D3vk 18C4vk 18E5vk 20E8vk 22E2vk 24A54vk 26F7Vk 27G8vk 31H9vk 34G3Vk 34D9Vk 43F5vk 73H10Vk 74H3Vk Table 2: Anti-IL-23p19 Mouse Leads - VH Sequences 2Dlvh 6B8VH 9D12VH 15C11vh 15F1vh 18D3vh 18C4vh 18E5vh 20E8vh 22E2vh 24A5vh 26F7VH 27G8vh 31H9vh 34G3VH 34D9VH7 43F5vh 73H10VH
  • Human framework sequences were selected for each of the mouse leads based on the framework homology, CDR structure, conserved canonical residues, conserved interface packing residues and other parameters.
  • Table 3 LIGHT CHAIN CDR sequences L-CDR1 L-CDR2 L-CDR3 18C4 RASQSISEYLH (SEQ ID NO:1) YASQSIS (SEQ ID NO:2) QNGHSFPFT (SEQ ID NO:3) 18E5 RASQSISDYLY (SEQ ID NO:4) FASQSIS (SEQ ID NO:5) QNGHSFPFT (SEQ ID NO:3) 18D3 RASQSISDYLY (SEQ ID NO:4) FASQSIS (SEQ ID NO:5) QNGHSFPFT (SEQ ID NO:3) 20E8 RASQSISEYLH (SEQ ID NO:1) YASQSIS (SEQ ID NO:2) QNGHSFPFT (SEQ ID NO:3) 22E2 RASQSISVYLH (SEQ ID NO:
  • IgG1KO k nock- o ut of effector functions
  • Leu234Ala Leu235Ala
  • Example 1 describes the humanization process in further detail. The results of such humanization resulted in humanized antibody sequences.
  • a representative number of humanized light chain and heavy chain variable regions derived from mouse antibody 6B8 are provided and shown in Tables 5 and 6.
  • An alignment between the humanized light chain and heavy chain variable regions derived from mouse antibody 6B8 and the chain and heavy chain variable regions from mouse antibody 6B8 is shown in Figure 1 .
  • Antibodies A, B, C and D have the heavy and light chain sequences shown in Table 7.
  • Table 5 Humanized 6B8-VK Sequences 6B8CVK-65 6B8CVK-66 6B8CVK-67 6B8CVK-78
  • Table 6 Humanized 6B8-VH Sequence 6B8CVH-02 6B8CVH-05 6B8CVH-36 6B8CVH-65
  • Table 7 Heavy and Light Chain DNA and Amino Acid Sequences for Antibodies A, B, C, and D
  • Antibody A IgK light Chain #66 IgG1 KO Heavy Chain #2
  • Antibody B IgK light Chain #66 (SEQ ID NO:173) (SEQ ID NO:174) IgGIKO Heavy Chain #5
  • Antibody C IgK light Chain #65 IgG1KO Heavy Chain #2 (SEQ ID NO:175) (SEQ ID NO:176)
  • Antibody D IgK light Chain #65 (SEQ ID NO:179) (SEQ ID NO
  • Antibodies A, B, C, and D Light chains and heavy chain variable regions of Antibodies A, B, C, and D are underlined in Table 7 above.
  • a humanized anti-IL-23p19 antibody of the present disclosure has at least one of the properties below. In a further aspect, a humanized anti-IL-23p19 antibody of the present disclosure has any combination of at least two, or at least 3, 4, 5, 6, 7, 8, 9, 10, or 11 of the properties below. In a further aspect, a humanized anti-IL-23p19 antibody of the present disclosure has all the properties below.
  • a humanized anti-IL-23p19 antibody of the present disclosure has reduced affinity to Fc receptor and therefore is predicted not to have activity in ADCC/CDC.
  • a humanized anti-IL-23p19 antibody of the present disclosure has at least one of the properties below. In a further aspect, a humanized anti-IL-23p19 antibody of the present disclosure has any combination of at least two, or at least 3, 4, 5, 6, 7, 8, 9, or 10 of the properties below. In a further aspect, a humanized anti-IL-23p19 antibody of the present disclosure has all the properties below.
  • a humanized antibody of the present disclosure has at least one of the following binding properties (properties A).
  • a humanized anti-IL-23p19 antibody of the present disclosure has any combination of at least two, or at least 3, of the properties below.
  • a humanized anti-IL-23p19 antibody of the present disclosure has all the properties below.
  • a humanized antibody of the present disclosure has a K D for human IL-23 ⁇ 1 pM (no shift in binding on-rate in 50% human serum) and no binding to human IL-12.
  • a humanized antibody of the present disclosure has at least one of the following functional properties (properties B).
  • a humanized anti-IL-23p19 antibody of the present disclosure has any combination of at least two, or at least 3, of the properties below.
  • a humanized anti-IL-23p19 antibody of the present disclosure has all the properties below.
  • a humanized antibody of the presentdisclosure has at least one of the following properties (properties C).
  • a humanized anti-IL-23p19 antibody of the present disclosure has any combination of at least two, or at least 3, of the properties below.
  • a humanized anti-IL-23p19 antibody of the present disclosure has all the properties below.
  • a humanized antibody of the present disclosure has at least one of the following properties (properties C).
  • a humanized anti-IL-23p19 antibody of the present invention has any combination of at least two of the properties below.
  • a humanized anti-IL-23p19 antibody of the present disclosure has all the properties below.
  • a humanized antibody of the present disclosure has at least one property A, at least one property B and at least one property C.
  • a humanized anti-IL-23p19 antibody of the present disclosure has any combination of at least two, or at least 3, of the properties A, B, and C.
  • the humanized antibody displays blocking activity, whereby it decreases the binding of IL-23 to IL-23 receptor by at least 45%, by at least 50%, by at least 55%, by at least 60%, by at least 65%, by at least 70%, by at least 75%, by at least 80%, by at least 85%, by at least 90%, or by at least 95%.
  • the ability of an antibody to block binding of IL-23 to the IL-23 receptor can be measured using competitive binding assays known in the art.
  • the blocking activity of an antibody can be measured by assessing the biological effects of IL-23, such as the production of IL-17 and IL-22 to determine if signaling mediated by the IL-23 receptor is inhibited.
  • the present disclosure provides a humanized anti-IL-23p19 antibody having favorable biophysical properties.
  • a humanized anti-IL-23p19 antibody of the present disclosure is present in at least 90% monomer form, or in at least 92% monomer form, or in at least 95% monomer form in a buffer.
  • a humanized anti-IL-23p19 antibody of the present disclosure remains in at least 90% monomer form, or in at least 92% monomer form, or in at least 95% monomer form in a buffer for one month or for four months.
  • a humanized antibody of the present disclosure is Antibody A, Antibody B, Antibody C or Antibody D.
  • the humanized antibody of the composition of the present invention comprises or consists of the light chain sequence of SEQ ID NO:174 and the heavy chain sequence of SEQ ID NO:176 (Antibody A).
  • a humanized antibody of the present disclosure comprises or consists of the light chain sequence of SEQ ID NO:174 and the heavy chain sequence of SEQ ID NO:178 (Antibody B).
  • a humanized antibody of the present disclosure comprises or consists of the light chain sequence of SEQ ID NO:180 and the heavy chain sequence of SEQ ID NO:176 (Antibody C).
  • a humanized antibody of the present disclosure comprises or consists of the light chain sequence of SEQ ID NO:180 and the heavy chain sequence of SEQ ID NO:178 (Antibody D).
  • the present disclosure provides an anti-IL-23p19 antibody that binds to human IL-23p19 at an epitope consisting of amino acid residues 108 to 126 and amino acid residues 137 to 151 of SEQ ID NO: 181.
  • the present disclosure provides an anti-IL-23p19 antibody or antigen-binding fragment thereof that competitively binds to human IL-23p19 with an antibody of the present invention, for example Antibody A, Antibody B, Antibody C or Antibody D described herein.
  • an antibody or antigen-binding fragment to competitively bind to IL-23p19 can be measured using competitive binding assays known in the art.
  • the humanized anti-IL-23p19 antibodies optionally include specific amino acid substitutions in the consensus or germline framework regions.
  • the specific substitution of amino acid residues in these framework positions can improve various aspects of antibody performance including binding affinity and/or stability, over that demonstrated in humanized antibodies formed by "direct swap" of CDRs or HVLs into the human germline framework regions.
  • the present disclosure describes other monoclonal antibodies with a light chain variable region having the amino acid sequence set forth in of SEQ ID NO: 84, 86, 88, 90, 91, 93, 95, 97, 99, 101, 103, 105, 107, 109, 111, 113, 115, 117 or 119.
  • the present disclosure describes other monoclonal antibodies with a heavy chain variable region having the amino acid sequence set forth in of SEQ ID NO: 121, 123, 125, 127, 128, 130, 132, 134, 136, 138, 140, 142, 144, 146, 148, 150, 152, 154 or 156 (see Tables 1 and 2 above).
  • the CDR sequence of these mouse antibodies are shown in Tables 3 and 4. Placing such CDRs into FRs of the human consensus heavy and light chain variable domains will yield useful humanized antibodies of the present invention.
  • the present disclosure provides monoclonal antibodies with the combinations of light chain variable and heavy chain variable regions of SEQ ID NO:84/121, 86/123, 88/125, 90/127, 91/128, 93/130, 95/132, 97/134, 99/136, 101/138, 103/140, 105/142, 107/144, 109/146, 111/148, 113/150, 115/152, 117/154 or 119/156.
  • Such variable regions can be combined with human constant regions.
  • the present disclosure describes other humanized antibodies with light chain variable region sequences having the amino acid sequence set forth in of SEQ ID NO:158, 160, 162 or 164.
  • the present disclosure describes other humanized antibodies with heavy chain variable region sequences having the amino acid sequence set forth in of SEQ ID NO:166, 168, 170 or 172 (see Tables 5 and 6 above). The CDR sequences of these antibodies are shown in Tables 3 and 4.
  • the present disclosure provides monoclonal antibodies with the combinations of light chain variable and heavy chain variable regions of SEQ ID NO: 160/166, 160/168, 158/166 or 158/168. Such variable regions can be combined with human constant regions.
  • the present disclosure relates to an anti-IL-23p19 antibody or antigen-binding fragment thereof comprising a humanized light chain variable domain comprising the CDRs of SEQ ID NO:160 and framework regions having an amino acid sequence at least 90% identical, at least 93% identical or at least 95% identical to the amino acid sequence of the framework regions of the variable domain light chain amino acid sequence of SEQ ID NO:160 and a humanized heavy chain variable domain comprising the CDRs of SEQ ID NO:166 and framework regions having an amino acid sequence at least 90% identical, at least 93% identical or at least 95% identical to the amino acid sequence of the framework regions of the variable domain heavy chain amino acid sequence of SEQ ID NO:166.
  • the anti-IL-23p19 antibody is a humanized monoclonal antibody.
  • the present disclosure relates to an anti-IL-23p19 antibody or antigen-binding fragment thereof comprising a humanized light chain variable domain comprising the CDRs of SEQ ID NO:160 and framework regions having an amino acid sequence at least 90% identical, at least 93% identical or at least 95% identical to the amino acid sequence of the framework regions of the variable domain light chain amino acid sequence of SEQ ID NO:160 and a humanized heavy chain variable domain comprising the CDRs of SEQ ID NO:168 and framework regions having an amino acid sequence at least 90% identical, at least 93% identical or at least 95% identical to the amino acid sequence of the framework regions of the variable domain heavy chain amino acid sequence of SEQ ID NO:168.
  • the anti-IL-23p19 antibody is a humanized monoclonal antibody.
  • the present disclosure relates to an anti-IL-23p19 antibody or antigen-binding fragment thereof comprising a humanized light chain variable domain comprising the CDRs of SEQ ID NO:158 and framework regions having an amino acid sequence at least 90% identical, at least 93% identical or at least 95% identical to the amino acid sequence of the framework regions of the variable domain light chain amino acid sequence of SEQ ID NO:158 and a humanized heavy chain variable domain comprising the CDRs of SEQ ID NO:166 and framework regions having an amino acid sequence at least 90% identical, at least 93% identical or at least 95% identical to the amino acid sequence of the framework regions of the variable domain heavy chain amino acid sequence of SEQ ID NO:166.
  • the anti-IL-23p19 antibody is a humanized monoclonal antibody.
  • the present disclosure relates to an anti-IL-23p19 antibody or antigen-binding fragment thereof comprising a humanized light chain variable domain comprising the CDRs of SEQ ID NO:158 and framework regions having an amino acid sequence at least 90% identical, at least 93% identical or at least 95% identical to the amino acid sequence of the framework regions of the variable domain light chain amino acid sequence of SEQ ID NO:158 and a humanized heavy chain variable domain comprising the CDRs of SEQ ID NO:168 and framework regions having an amino acid sequence at least 90% identical, at least 93% identical or at least 95% identical to the amino acid sequence of the framework regions of the variable domain heavy chain amino acid sequence of SEQ ID NO:168.
  • the anti-IL-23p19 antibody is a humanized monoclonal antibody.
  • the humanized anti-IL-23p19 antibodies disclosed herein comprise at least a heavy or a light chain variable domain comprising the CDRs or HVLs of the murine monoclonal antibodies or humanized antibodies as shown in Tables 1 through 6 above and the FRs of the human germline heavy and light chain variable domains.
  • the present disclosure provides an anti-IL-23p19 antibody or antigen-binding fragment thereof comprising a light chain CDR1 (L-CDR1) sequence of SEQ ID NO:1, 4, 6, 7, 8, 11, 15, 18, 19, 22, 27 or 30; a light chain CDR2 (L-CDR2) sequence of SEQ ID NO:2, 5, 9, 12, 16, 20, 23, 25, 28 or 31; a light chain CDR3 (L-CDR3) sequence of SEQ ID NO:3, 10, 13, 14, 17, 21, 24, 26, 29, or 32; a heavy chain CDR1 (H-CDR1) sequence of SEQ ID NO:33, 36, 38, 40, 43, 45, 48, 51, 54, 57, 60, 63, 66, 67, 68, 69, 77 or 80; a heavy chain CDR2 (H-CDR2) sequence of SEQ ID NO:34, 39, 41, 46, 49, 52, 55, 58, 61, 64, 70, 72, 73
  • the anti-IL-23p19 antibody or antigen-binding fragment thereof comprises a light chain variable region comprising a L-CDR1 listed above, a L-CDR2 listed above and a L-CDR3 listed above, and a heavy chain variable region comprising a H-CDR1 listed above, a H-CDR2 listed above and a H-CDR3 listed above.
  • an anti-IL-23p19 antibody or antigen-binding fragment thereof comprising:
  • the anti-IL-23p19 antibody or antigen-binding fragment thereof comprises a light chain variable region comprising a L-CDR1, L-CDR2 and L-CDR3 combination listed above, and a heavy chain variable region comprising a H-CDR1, H-CDR2 and H-CDR3 combination listed above.
  • chimeric antibodies with switched CDR regions i.e., for example switching one or two CDRs of one of the mouse antibodies or humanized antibody derived therefrom with the analogous CDR from another mouse antibody or humanized antibody derived therefrom
  • switching one or two CDRs of one of the mouse antibodies or humanized antibody derived therefrom with the analogous CDR from another mouse antibody or humanized antibody derived therefrom may yield useful antibodies.
  • the humanized anti-IL-23p19 antibody is an antibody fragment.
  • Various antibody fragments have been generally discussed above and there are techniques that have been developed for the production of antibody fragments. Fragments can be derived via proteolytic digestion of intact antibodies (see, e.g., Morimoto et al., 1992, Journal of Biochemical and Biophysical Methods 24:107-117 ; and Brennan et al., 1985, Science 229:81 ). Alternatively, the fragments can be produced directly in recombinant host cells. For example, Fab'-SH fragments can be directly recovered from E.
  • the present disclosure provides antibody fragments comprising the CDRs described herein, in particular one of the combinations of L-CDR1, L-CDR2, L-CDR3, H-CDR1, H-CDR2 and H-CDR3 described herein.
  • the present disclosure provides antibody fragments comprising the variable regions described herein, for example one of the combinations of light chain variable regions and heavy chain variable regions described herein.
  • Certain reference aspects include an F(ab') 2 fragment of a humanized anti-IL-23p19 antibody comprise a light chain sequence of any of SEQ ID NO: 174 or 180 in combination with a heavy chain sequence of SEQ ID NO: 176 or 178.
  • Such reference aspects can include an intact antibody comprising such an F(ab') 2 .
  • the antibody or antibody fragment includes a constant region that mediates effector function.
  • the constant region can provide antibody-dependent cellular cytotoxicity (ADCC), antibody-dependent cellular phagocytosis (ADCP) and/or complement-dependent cytotoxicity (CDC) responses against an IL-23 expressing target cell.
  • the effector domain(s) can be, for example, an Fc region of an Ig molecule.
  • the effector domain of an antibody can be from any suitable vertebrate animal species and isotypes.
  • the isotypes from different animal species differ in the abilities to mediate effector functions.
  • the ability of human immunoglobulin to mediate CDC and ADCC/ADCP is generally in the order of IgM ⁇ IgG 1 ⁇ IgG 3 >IgG 2 >IgG 4 and IgG 1 ⁇ IgG 3 >IgG 2 /IgM/IgG 4 , respectively.
  • Murine immunoglobulins mediate CDC and ADCC/ADCP generally in the order of murine IgM ⁇ IgG 3 >>IgG 2b >IgG 2a >>IgG 1 and IgG 2b >IgG 2a >IgG 1 >>IgG 3 , respectively.
  • murine IgG 2a mediates ADCC while both murine IgG 2a and IgM mediate CDC.
  • the humanized anti-IL-23p19 antibodies and agents can include modifications of the humanized anti-IL-23p19 antibody or antigen-binding fragment thereof. For example, it may be desirable to modify the antibody with respect to effector function, so as to enhance the effectiveness of the antibody in treating cancer.
  • One such modification is the introduction of cysteine residue(s) into the Fc region, thereby allowing interchain disulfide bond formation in this region.
  • the homodimeric antibody thus generated can have improved internalization capability and/or increased complement-mediated cell killing and/or antibody-dependent cellular cytotoxicity (ADCC). See, for example, Caron et al., 1992, J. Exp Med. 176:1191-1195 ; and Shopes, 1992, J. Immunol.
  • Homodimeric antibodies having enhanced anti-tumor activity can also be prepared using heterobifunctional cross-linkers as described in Wolff et al., 1993, Cancer Research 53: 2560-2565 .
  • an antibody can be engineered to contain dual Fc regions, enhancing complement lysis and ADCC capabilities of the antibody. See Stevenson et al., 1989, Anti-Cancer Drug Design 3: 219-230 .
  • Antibodies with improved ability to support ADCC have been generated by modifying the glycosylation pattern of their Fc region. This is possible since antibody glycosylation at the asparagine residue, N297, in the C H2 domain is involved in the interaction between IgG and Fc ⁇ receptors prerequisite to ADCC.
  • Host cell lines have been engineered to express antibodies with altered glycosylation, such as increased bisecting N-acetylglucosamine or reduced fucose. Fucose reduction provides greater enhancement to ADCC activity than does increasing the presence of bisecting N-acetylglucosamine.
  • enhancement of ADCC by low fucose antibodies is independent of the Fc ⁇ RIIIa V/F polymorphism.
  • Modifying the amino acid sequence of the Fc region of antibodies is an alternative to glycosylation engineering to enhance ADCC.
  • the binding site on human IgG 1 for Fc ⁇ receptors has been determined by extensive mutational analysis. This led to the generation of humanized IgG 1 antibodies with Fc mutations that increase the binding affinity for Fc ⁇ RIIIa and enhance ADCC in vitro. Additionally, Fc variants have been obtained with many different permutations of binding properties, e.g., improved binding to specific FcyR receptors with unchanged or diminished binding to other FcyR receptors.
  • immunoconjugates comprising the humanized antibody or fragments thereof conjugated to a cytotoxic agent such as a chemotherapeutic agent, a toxin (e.g., an enzymatically active toxin of bacterial, fungal, plant, or animal origin, or fragments thereof), or a radioactive isotope (i.e., a radioconjugate).
  • a cytotoxic agent such as a chemotherapeutic agent, a toxin (e.g., an enzymatically active toxin of bacterial, fungal, plant, or animal origin, or fragments thereof), or a radioactive isotope (i.e., a radioconjugate).
  • Enzymatically active toxins and fragments thereof that can be used to form useful immunoconjugates include diphtheria A chain, nonbinding active fragments of diphtheria toxin, exotoxin A chain (from Pseudomonas aeruginosa), ricin A chain, abrin A chain, modeccin A chain, alpha-sarcin, Aleurites fordii proteins, dianthin proteins, Phytolaca americana proteins (PAPI, PAPII, and PAP-S), Momordica charantia inhibitor, curcin, crotin, Sapaonaria officinalis inhibitor, gelonin, mitogellin, restrictocin, phenomycin, enomycin, the tricothecenes, and the like.
  • a variety of radionuclides are available for the production of radioconjugated humanized anti-IL-23p19 antibodies. Examples include 212 Bi,
  • Conjugates of the humanized anti-IL-23p19 antibody and cytotoxic or chemotherapeutic agent can be made by known methods, using a variety of bifunctional protein coupling agents such as N-succinimidyl-3-(2-pyridyldithiol) propionate (SPDP), iminothiolane (IT), bifunctional derivatives of imidoesters (such as dimethyl adipimidate HCL), active esters (such as disuccinimidyl suberate), aldehydes (such as glutareldehyde), bis-azido compounds (such as bis (p-azidobenzoyl) hexanediamine), bis-diazonium derivatives (such as bis-(p-diazoniumbenzoyl)-ethylenediamine), diisocyanates (such as toluene 2,6-diisocyanate), and bis-active fluorine compounds (such as 1,5-difluoro-2,4-dinitro
  • a ricin immunotoxin can be prepared as described in Vitetta et al., 1987, Science 238:1098 .
  • Carbon-14-labeled 1-isothiocyanatobenzyl-3-methyldiethylene triaminepentaacetic acid (MX-DTPA) is an exemplary chelating agent for conjugation of radionucleotide to the antibody.
  • Conjugates also can be formed with a cleavable linker.
  • the humanized anti-IL-23p19 antibodies disclosed herein can also be formulated as immunoliposomes.
  • Liposomes containing the antibody are prepared by methods known in the art, such as described in Epstein et al., 1985, Proc. Natl. Acad. Sci. USA 82:3688 ; Hwang et al., 1980, Proc. Natl. Acad. Sci. USA 77:4030 ; and U.S. Pat. Nos. 4,485,045 and 4,544,545 .
  • Liposomes having enhanced circulation time are disclosed, for example, in U.S. Pat. No. 5,013,556 .
  • Particularly useful liposomes can be generated by the reverse phase evaporation method with a lipid composition comprising phosphatidylcholine, cholesterol and PEG-derivatized phosphatidylethanolamine (PEG-PE). Liposomes are extruded through filters of defined pore size to yield liposomes with the desired diameter.
  • Fab' fragments of an antibody disclosed herein can be conjugated to the liposomes as described in Martin et al., 1982, J. Biol. Chem. 257:286-288 via a disulfide interchange reaction.
  • a chemotherapeutic agent such as doxorubicin is optionally contained within the liposome. See, e.g., Gabizon et al., 1989, J. National Cancer Inst. 81(19):1484 .
  • the antibodies described and disclosed herein can also be used in ADEPT (Antibody-Directed Enzyme Prodrug Therapy) procedures by conjugating the antibody to a prodrug-activating enzyme that converts a prodrug (e.g., a peptidyl chemotherapeutic agent), to an active anti-cancer drug.
  • ADEPT Antibody-Directed Enzyme Prodrug Therapy
  • a prodrug e.g., a peptidyl chemotherapeutic agent
  • the enzyme component of the immunoconjugate useful for ADEPT is an enzyme capable of acting on a prodrug in such a way so as to covert it into its more active, cytotoxic form.
  • Specific enzymes that are useful in ADEPT include, but are not limited to, alkaline phosphatase for converting phosphate-containing prodrugs into free drugs; arylsulfatase for converting sulfate-containing prodrugs into free drugs; cytosine deaminase for converting non-toxic 5-fluorocytosine into the anti-cancer drug, 5-fluorouracil; proteases, such as serratia protease, thermolysin, subtilisin, carboxypeptidases, and cathepsins (such as cathepsins B and L), for converting peptide-containing prodrugs into free drugs; D-alanylcarboxypeptidases, for converting prodrugs containing D-amino acid substituents; carbohydrate-cleaving enzymes such as ⁇ -galactosidase and neuraminidase for converting glycosylated prodrugs into free drugs; ⁇ -lactamase for converting drugs
  • antibodies having enzymatic activity can be used to convert the prodrugs into free active drugs (see, for example, Massey, 1987, Nature 328: 457-458 ).
  • Antibody-abzyme conjugates can be prepared by known methods for delivery of the abzyme to a tumor cell population, for example, by covalently binding the enzyme to the humanized anti-IL-23p19antibody/heterobifunctional crosslinking reagents discussed above.
  • fusion proteins comprising at least the antigen binding region of an antibody disclosed herein linked to at least a functionally active portion of an enzyme as described above can be constructed using recombinant DNA techniques (see, e.g., Neuberger et al., 1984, Nature 312:604-608 ).
  • a humanized anti-IL-23p19 antibody fragment rather than an intact antibody, to increase tissue penetration, for example. It may be desirable to modify the antibody fragment in order to increase its serum half life. This can be achieved, for example, by incorporation of a salvage receptor binding epitope into the antibody fragment.
  • the appropriate region of the antibody fragment can be altered (e.g., mutated), or the epitope can be incorporated into a peptide tag that is then fused to the antibody fragment at either end or in the middle, for example, by DNA or peptide synthesis. See, e.g., WO 96/32478 .
  • covalent modifications of the humanized anti-IL-23p19 antibody are also included.
  • Covalent modifications include modification of cysteinyl residues, histidyl residues, lysinyl and amino-terminal residues, arginyl residues, tyrosyl residues, carboxyl side groups (aspartyl or glutamyl), glutaminyl and asparaginyl residues, or seryl, or threonyl residues.
  • Another type of covalent modification involves chemically or enzymatically coupling glycosides to the antibody. Such modifications may be made by chemical synthesis or by enzymatic or chemical cleavage of the antibody, if applicable.
  • Other types of covalent modifications of the antibody can be introduced into the molecule by reacting targeted amino acid residues of the antibody with an organic derivatizing agent that is capable of reacting with selected side chains or the amino- or carboxy-terminal residues.
  • Another type of useful covalent modification comprises linking the antibody to one of a variety of nonproteinaceous polymers, e.g., polyethylene glycol, polypropylene glycol, or polyoxyalkylenes, in the manner set forth in one or more of U.S. Pat. No. 4,640,835 , U.S. Pat. No. 4,496,689 , U.S. Pat. No. 4,301,144 , U.S. Pat. No. 4,670,417 , U.S. Pat. No. 4,791,192 and U.S. Pat. No. 4,179,337 .
  • nonproteinaceous polymers e.g., polyethylene glycol, polypropylene glycol, or polyoxyalkylenes
  • Amino acid sequence variants of the anti-IL-23p19 antibody can be prepared by introducing appropriate nucleotide changes into the anti-IL-23p19 antibody DNA, or by peptide synthesis.
  • Such variants include, for example, deletions from, and/or insertions into and/or substitutions of, residues within the amino acid sequences of the anti-IL-23p19 antibodies of the examples herein. Any combination of deletions, insertions, and substitutions is made to arrive at the final construct, provided that the final construct possesses the desired characteristics.
  • the amino acid changes also may alter posttranslational processes of the humanized or variant anti-IL-23p19 antibody, such as changing the number or position of glycosylation sites.
  • a useful method for identification of certain residues or regions of the anti-IL-23p19 antibody that are preferred locations for mutagenesis is called “alanine scanning mutagenesis," as described by Cunningham and Wells (Science, 244:1081-1085 (1989 )).
  • a residue or group of target residues are identified (e.g., charged residues such as arg, asp, his, lys, and glu) and replaced by a neutral or negatively charged amino acid (typically alanine) to affect the interaction of the amino acids with IL-23p19 antigen.
  • Those amino acid locations demonstrating functional sensitivity to the substitutions then are refined by introducing further or other variants at, or for, the sites of substitution.
  • the site for introducing an amino acid sequence variation is predetermined, the nature of the mutation per se need not be predetermined.
  • alanine scanning or random mutagenesis is conducted at the target codon or region and the expressed anti-IL-23p19 antibody variants are screened for the desired activity.
  • Amino acid sequence insertions include amino- and/or carboxyl-terminal fusions ranging in length from one residue to polypeptides containing a hundred or more residues, as well as intrasequence insertions of single or multiple amino acid residues.
  • terminal insertions include an anti-IL-23p19 antibody fused to an epitope tag.
  • Other insertional variants of the anti-IL-23p19 antibody molecule include a fusion to the N- or C-terminus of the anti-IL-23p19 antibody of an enzyme or a polypeptide which increases the serum half-life of the antibody.
  • variants are an amino acid substitution variant. These variants have at least one amino acid residue in the anti-IL-23p19 antibody molecule removed and a different residue inserted in its place.
  • the sites of greatest interest for substitutional mutagenesis include the hypervariable regions, but FR alterations are also contemplated. Conservative substitutions are shown in Table 5 under the heading of "preferred substitutions". If such substitutions result in a change in biological activity, then more substantial changes, denominated "exemplary substitutions", or as further described below in reference to amino acid classes, may be introduced and the products screened.
  • the biological properties of the antibody can be accomplished by selecting substitutions that differ significantly in their effect on maintaining (a) the structure of the polypeptide backbone in the area of the substitution, for example, as a sheet or helical conformation, (b) the charge or hydrophobicity of the molecule at the target site, or (c) the bulk of the side chain.
  • Naturally occurring residues are divided into groups based on common side-chain properties:
  • Non-conservative substitutions will entail exchanging a member of one of these classes for another class.
  • cysteine residue not involved in maintaining the proper conformation of the humanized or variant anti-IL-23p19 antibody also may be substituted, generally with serine, to improve the oxidative stability of the molecule, prevent aberrant crosslinking, or provide for established points of conjugation to a cytotoxic or cytostatic compound.
  • cysteine bond(s) may be added to the antibody to improve its stability (particularly where the antibody is an antibody fragment such as an Fv fragment).
  • a type of substitutional variant involves substituting one or more hypervariable region residues of a parent antibody (e.g., a humanized or human antibody).
  • a parent antibody e.g., a humanized or human antibody
  • the resulting variant(s) selected for further development will have improved biological properties relative to the parent antibody from which they are generated.
  • a convenient way for generating such substitutional variants is affinity maturation using phage display. Briefly, several hypervariable region sites (e.g., 6-7 sites) are mutated to generate all possible amino substitutions at each site.
  • the antibody variants thus generated are displayed in a monovalent fashion from filamentous phage particles as fusions to the gene III product of M13 packaged within each particle. The phage-displayed variants are then screened for their biological activity (e.g., binding affinity).
  • alanine scanning mutagenesis can be performed to identify hypervariable region residues contributing significantly to antigen binding.
  • Such contact residues and neighboring residues are candidates for substitution according to the techniques elaborated herein.
  • Another type of amino acid variant of the antibody alters the original glycosylation pattern of the antibody.
  • altering is meant deleting one or more carbohydrate moieties found in the antibody, and/or adding one or more glycosylation sites that are not present in the antibody.
  • glycosylations sites it may be desirable to modify the antibodies of the invention to add glycosylations sites.
  • Glycosylation of antibodies is typically either N-linked or O-linked.
  • N-linked refers to the attachment of the carbohydrate moiety to the side chain of an asparagine residue.
  • the tripeptide sequences asparagine-X-serine and asparagine-X-threonine, where X is any amino acid except proline, are the recognition sequences for enzymatic attachment of the carbohydrate moiety to the asparagine side chain.
  • the presence of either of these tripeptide sequences in a polypeptide creates a potential glycosylation site.
  • O-linked glycosylation refers to the attachment of one of the sugars N-aceylgalactosamine, galactose, or xylose to a hydroxyamino acid, most commonly serine or threonine, although 5-hydroxyproline or 5-hydroxylysine may also be used.
  • a given protein e.g., an antibody
  • the amino acid sequence of the protein is engineered to contain one or more of the above-described tripeptide sequences (for N-linked glycosylation sites).
  • the alteration may also be made by the addition of, or substitution by, one or more serine or threonine residues to the sequence of the original antibody (for O-linked glycosylation sites).
  • Nucleic acid molecules encoding amino acid sequence variants of the anti-IL-23p19 antibody are prepared by a variety of methods known in the art. These methods include, but are not limited to, isolation from a natural source (in the case of naturally occurring amino acid sequence variants) or preparation by oligonucleotide-mediated (or sitedirected) mutagenesis, PCR mutagenesis, and cassette mutagenesis of an earlier prepared variant or a non-variant version of the anti-IL-23p19 antibody.
  • isolated polynucleotides that comprise a sequence encoding a humanized anti-IL-23p19 antibody, vectors, and host cells comprising the polynucleotides, and recombinant techniques for production of the humanized antibody.
  • the isolated polynucleotides can encode any desired form of the anti-IL-23p19 antibody including, for example, full length monoclonal antibodies, Fab, Fab', F(ab') 2 , and Fv fragments, diabodies, linear antibodies, single-chain antibody molecules, and multispecific antibodies formed from antibody fragments.
  • Some reference aspects include isolated polynucleotides comprising sequences that encode the light chain variable region of an antibody or antibody fragment having the amino acid sequence of any of SEQ ID NO: SEQ ID NO:84, 86, 88, 90, 91, 93, 95, 97, 99,101, 103, 105, 107, 109, 111, 113, 115, 117 or 119.
  • Exemplary polynucleotide sequences encoding such amino acid sequences are SEQ ID NO:83, 85, 87, 89, 92, 94, 96, 98, 100, 102, 104, 106, 108, 110, 112, 114, 116 and 118.
  • Some embodiments include isolated polynucleotides comprising sequences that encode the heavy chain variable region of an antibody or antibody fragment having the amino acid sequence of SEQ ID NO:121, 123, 125, 127, 128, 130, 132, 134, 136, 138, 140, 142, 144, 146, 148, 150, 152, 154 or 156.
  • Exemplary polynucleotide sequences encoding such amino acid sequences are SEQ ID NO: 120, 122, 124, 126, 129, 131, 133, 135, 137, 139, 141, 143, 145, 147, 149, 151, 153 or 155.
  • Some reference aspects include isolated polynucleotides comprising sequences that encode the light chain variable region of an antibody or antibody fragment having the amino acid sequence of any of SEQ ID NO:158, 160, 162 or 164.
  • Exemplary polynucleotide sequences encoding such amino acid sequences are SEQ ID NO:157, 159, 161 or 163.
  • Some embodiments include isolated polynucleotides comprising sequences that encode the heavy chain variable region of an antibody or antibody fragment having the amino acid sequence of SEQ ID NO: 166, 168, 170 or 172.
  • Exemplary polynucleotide sequences encoding such amino acid sequences are SEQ ID NO: 165, 167, 169 or 171.
  • Some reference aspects include isolated polynucleotides comprising sequences that encode the light chain of an antibody having the amino acid sequence of any of SEQ ID NO:174 or 180. Exemplary polynucleotide sequences encoding such amino acid sequences are SEQ ID NO:173 or 179. Some reference aspects include isolated polynucleotides comprising sequences that encode the heavy chain of an antibody having the amino acid sequence of SEQ ID NO:176 or 178. Exemplary polynucleotide sequences encoding such amino acid sequences are SEQ ID NO:175 or 177.
  • the isolated polynucleotide sequence(s) encodes an antibody or antibody fragment having a light chain and a heavy chain variable region comprising the amino acid sequences of SEQ ID NO:174 and SEQ ID NO:176, respectively; SEQ ID NO:174 and SEQ ID NO:178, respectively; SEQ ID NO:180 and SEQ ID NO:176, respectively; SEQ ID NO:180 and SEQ ID NO:178, respectively.
  • Exemplary polynucleotide sequences encoding such amino acid sequences are SEQ ID NO: 173 and 175, respectively, SEQ ID NO: 173 and 177, respectively, SEQ ID NO: 179 and 175, respectively, SEQ ID NO: 179 and 177, respectively.
  • polynucleotide(s) that comprise a sequence encoding a humanized anti-IL-23p19 antibody or a fragment or chain thereof can be fused to one or more regulatory or control sequence, as known in the art, and can be contained in suitable expression vectors or host cell as known in the art.
  • Each of the polynucleotide molecules encoding the heavy or light chain variable domains can be independently fused to a polynucleotide sequence encoding a constant domain, such as a human constant domain, enabling the production of intact antibodies.
  • polynucleotides, or portions thereof can be fused together, providing a template for production of a single chain antibody.
  • a polynucleotide encoding the antibody is inserted into a replicable vector for cloning (amplification of the DNA) or for expression.
  • a replicable vector for cloning amplification of the DNA
  • vectors for expressing the recombinant antibody are available.
  • the vector components generally include, but are not limited to, one or more of the following: a signal sequence, an origin of replication, one or more marker genes, an enhancer element, a promoter, and a transcription termination sequence.
  • the humanized anti-IL-23p19 antibodies can also be produced as fusion polypeptides, in which the antibody is fused with a heterologous polypeptide, such as a signal sequence or other polypeptide having a specific cleavage site at the amino terminus of the mature protein or polypeptide.
  • a heterologous polypeptide such as a signal sequence or other polypeptide having a specific cleavage site at the amino terminus of the mature protein or polypeptide.
  • the heterologous signal sequence selected is typically one that is recognized and processed (i.e., cleaved by a signal peptidase) by the host cell.
  • the signal sequence can be substituted by a prokaryotic signal sequence.
  • the signal sequence can be, for example, alkaline phosphatase, penicillinase, lipoprotein, heat-stable enterotoxin II leaders, and the like.
  • yeast secretion the native signal sequence can be substituted, for example, with a leader sequence obtained from yeast invertase alpha-factor (including Saccharomyces and Kluyveromyces ⁇ -factor leaders), acid phosphatase, C. albicans glucoamylase, or the signal described in WO90/13646 .
  • yeast invertase alpha-factor including Saccharomyces and Kluyveromyces ⁇ -factor leaders
  • acid phosphatase C. albicans glucoamylase
  • mammalian signal sequences as well as viral secretory leaders for example, the herpes simplex gD signal, can be used.
  • the DNA for such precursor region is ligated in reading frame to DNA encoding the humanized anti-IL-23p19 antibody.
  • Expression and cloning vectors contain a nucleic acid sequence that enables the vector to replicate in one or more selected host cells.
  • this sequence is one that enables the vector to replicate independently of the host chromosomal DNA, and includes origins of replication or autonomously replicating sequences.
  • origins of replication or autonomously replicating sequences are well known for a variety of bacteria, yeast, and viruses.
  • the origin of replication from the plasmid pBR322 is suitable for most Gram-negative bacteria, the 2- ⁇ . plasmid origin is suitable for yeast, and various viral origins (SV40, polyoma, adenovirus, VSV, and BPV) are useful for cloning vectors in mammalian cells.
  • the origin of replication component is not needed for mammalian expression vectors (the SV40 origin may typically be used only because it contains the early promoter).
  • Expression and cloning vectors may contain a gene that encodes a selectable marker to facilitate identification of expression.
  • Typical selectable marker genes encode proteins that confer resistance to antibiotics or other toxins, e.g., ampicillin, neomycin, methotrexate, or tetracycline, or alternatively, are complement auxotrophic deficiencies, or in other alternatives supply specific nutrients that are not present in complex media, e.g., the gene encoding D-alanine racemase for Bacilli.
  • One example of a selection scheme utilizes a drug to arrest growth of a host cell. Those cells that are successfully transformed with a heterologous gene produce a protein conferring drug resistance and thus survive the selection regimen. Examples of such dominant selection use the drugs neomycin, mycophenolic acid, and hygromycin.
  • Common selectable markers for mammalian cells are those that enable the identification of cells competent to take up a nucleic acid encoding a humanized anti-IL-23p19 antibody, such as DHFR (dihydrofolate reductase), thymidine kinase, metallothionein-I and -II (such as primate metallothionein genes), adenosine deaminase, ornithine decarboxylase, and the like.
  • DHFR dihydrofolate reductase
  • adenosine deaminase ornithine decarboxylase
  • Cells transformed with the DHFR selection gene are first identified by culturing all of the transformants in a culture medium that contains methotrexate (Mtx), a competitive antagonist of DHFR.
  • host cells can be selected by cell growth in medium containing a selection agent for the selectable marker such as an aminoglycosidic antibiotic, e.g., kanamycin, neomycin, or G418. See, e.g., U.S. Pat. No. 4,965,199 .
  • selectable marker such as an aminoglycosidic antibiotic, e.g., kanamycin, neomycin, or G418. See, e.g., U.S. Pat. No. 4,965,199 .
  • the TRP1 gene present in the yeast plasmid YRp7 can be used as a selectable marker.
  • the TRP1 gene provides a selection marker for a mutant strain of yeast lacking the ability to grow in tryptophan, for example, ATCC No. 44076 or PEP4-1 ( Jones, 1977, Genetics 85:12 ).
  • the presence of the trp1 lesion in the yeast host cell genome then provides an effective environment for detecting transformation by growth in the absence of tryptophan.
  • Leu2p-deficient yeast strains such as ATCC 20,622 and 38,626 are complemented by known plasmids bearing the LEU2 gene.
  • vectors derived from the 1.6 ⁇ m circular plasmid pKD1 can be used for transformation of Kluyveromyces yeasts.
  • an expression system for largescale production of recombinant calf chymosin was reported for K. lactis ( Van den Berg, 1990, Bio/Technology 8:135 ).
  • Stable multi-copy expression vectors for secretion of mature recombinant human serum albumin by industrial strains of Kluyveromyces have also been disclosed ( Fleer et al., 1991, Bio/Technology 9:968-975 ).
  • Expression and cloning vectors usually contain a promoter that is recognized by the host organism and is operably linked to the nucleic acid molecule encoding an anti-IL-23p19 antibody or polypeptide chain thereof.
  • Promoters suitable for use with prokaryotic hosts include phoA promoter, ⁇ -lactamase and lactose promoter systems, alkaline phosphatase, tryptophan (trp) promoter system, and hybrid promoters such as the tac promoter. Other known bacterial promoters are also suitable. Promoters for use in bacterial systems also will contain a Shine-Dalgarno (S.D.) sequence operably linked to the DNA encoding the humanized anti-IL-23p19 antibody.
  • S.D. Shine-Dalgarno
  • eukaryotic promoter sequences are known. Virtually all eukaryotic genes have an AT-rich region located approximately 25 to 30 bases upstream from the site where transcription is initiated. Another sequence found 70 to 80 bases upstream from the start of transcription of many genes is a CNCAAT region where N may be any nucleotide. At the 3' end of most eukaryotic genes is an AATAAA sequence that may be the signal for addition of the poly A tail to the 3' end of the coding sequence. All of these sequences are suitably inserted into eukaryotic expression vectors.
  • suitable promoting sequences for use with yeast hosts include the promoters for 3-phosphoglycerate kinase or other glycolytic enzymes, such as enolase, glyceraldehyde-3-phosphate dehydrogenase, hexokinase, pyruvate decarboxylase, phosphofructokinase, glucose-6-phosphate isomerase, 3-phosphoglycerate mutase, pyruvate kinase, triosephosphate isomerase, phosphoglucose isomerase, and glucokinase.
  • 3-phosphoglycerate kinase or other glycolytic enzymes such as enolase, glyceraldehyde-3-phosphate dehydrogenase, hexokinase, pyruvate decarboxylase, phosphofructokinase, glucose-6-phosphate isomerase, 3-phosphoglycerate mutase, pyruvate
  • Inducible promoters have the additional advantage of transcription controlled by growth conditions. These include yeast promoter regions for alcohol dehydrogenase 2, isocytochrome C, acid phosphatase, derivative enzymes associated with nitrogen metabolism, metallothionein, glyceraldehyde-3-phosphate dehydrogenase, and enzymes responsible for maltose and galactose utilization. Suitable vectors and promoters for use in yeast expression are further described in EP 73,657 . Yeast enhancers also are advantageously used with yeast promoters.
  • Humanized anti-IL-23p19 antibody transcription from vectors in mammalian host cells is controlled, for example, by promoters obtained from the genomes of viruses such as polyoma virus, fowlpox virus, adenovirus (such as Adenovirus 2), bovine papilloma virus, avian sarcoma virus, cytomegalovirus, a retrovirus, hepatitis-B virus and Simian Virus 40 (SV40), from heterologous mammalian promoters, e.g., the actin promoter or an immunoglobulin promoter, or from heat-shock promoters, provided such promoters are compatible with the host cell systems.
  • viruses such as polyoma virus, fowlpox virus, adenovirus (such as Adenovirus 2), bovine papilloma virus, avian sarcoma virus, cytomegalovirus, a retrovirus, hepatitis-B virus and Simian Virus 40
  • the early and late promoters of the SV40 virus are conveniently obtained as an SV40 restriction fragment that also contains the SV40 viral origin of replication.
  • the immediate early promoter of the human cytomegalovirus is conveniently obtained as a HindIII E restriction fragment.
  • a system for expressing DNA in mammalian hosts using the bovine papilloma virus as a vector is disclosed in U.S. Pat. No. 4,419,446 . A modification of this system is described in U.S. Pat. No. 4,601,978 .
  • Rous sarcoma virus long terminal repeat can be used as the promoter.
  • enhancer sequence Another useful element that can be used in a recombinant expression vector is an enhancer sequence, which is used to increase the transcription of a DNA encoding a humanized anti-IL-23p19 antibody by higher eukaryotes.
  • enhancer sequences are now known from mammalian genes (e.g., globin, elastase, albumin, ⁇ -fetoprotein, and insulin).
  • an enhancer from a eukaryotic cell virus is used. Examples include the SV40 enhancer on the late side of the replication origin (bp 100-270), the cytomegalovirus early promoter enhancer, the polyoma enhancer on the late side of the replication origin, and adenovirus enhancers.
  • the enhancer may be spliced into the vector at a position 5' or 3' to the humanized anti-IL-23p19 antibody-encoding sequence, but is preferably located at a site 5' from the promoter.
  • Expression vectors used in eukaryotic host cells can also contain sequences necessary for the termination of transcription and for stabilizing the mRNA. Such sequences are commonly available from the 5' and, occasionally 3', untranslated regions of eukaryotic or viral DNAs or cDNAs. These regions contain nucleotide segments transcribed as polyadenylated fragments in the untranslated portion of the mRNA encoding anti-IL-23p19 antibody.
  • One useful transcription termination component is the bovine growth hormone polyadenylation region. See WO94/11026 and the expression vector disclosed therein.
  • humanized anti-IL-23p19 antibodies can be expressed using the CHEF system. (See, e.g., U.S. Pat. No. 5,888,809 ; the disclosure of which is incorporated by reference herein.)
  • Suitable host cells for cloning or expressing the DNA in the vectors herein are the prokaryote, yeast, or higher eukaryote cells described above.
  • Suitable prokaryotes for this purpose include eubacteria, such as Gram-negative or Gram-positive organisms, for example, Enterobacteriaceae such as Escherichia, e.g., E. coli, Enterobacter, Erwinia, Klebsiella, Proteus, Salmonella, e.g., Salmonella typhimurium, Serratia, e.g., Serratia marcescans, and Shigella, as well as Bacilli such as B. subtilis and B. licheniformis (e.g., B.
  • E. coli 294 ATCC 31,446
  • E. coli B E. coli X1776
  • E. coli W3110 ATCC 27,325
  • eukaryotic microbes such as filamentous fungi or yeast are suitable cloning or expression hosts for humanized anti-IL-23p19antibody-encoding vectors.
  • Saccharomyces cerevisiae or common baker's yeast, is the most commonly used among lower eukaryotic host microorganisms.
  • a number of other genera, species, and strains are commonly available and useful herein, such as Schizosaccharomyces pombe; Kluyveromyces hosts such as, e.g., K. lactis, K. fragilis (ATCC 12,424), K. bulgaricus (ATCC 16,045), K. wickeramii (ATCC 24,178), K.
  • waltii ATCC 56,500
  • K. drosophilarum ATCC 36,906
  • K. thermotolerans K. marxianus
  • yarrowia EP 402,226
  • Pichia pastors EP 183,070
  • Candida Trichoderma reesia
  • Neurospora crassa Schwanniomyces such as Schwanniomyces occidentalis
  • filamentous fungi such as, e.g., Neurospora, Penicillium, Tolypocladium, and Aspergillus hosts such as A. nidulans and A. niger.
  • Suitable host cells for the expression of glycosylated humanized anti-IL-23p19 antibody are derived from multicellular organisms.
  • invertebrate cells include plant and insect cells, including, e.g., numerous baculoviral strains and variants and corresponding permissive insect host cells from hosts such as Spodoptera frugiperda (caterpillar), Aedes aegypti (mosquito), Aedes albopictus (mosquito), Drosophila melanogaster (fruitfly), and Bombyx mori (silk worm).
  • a variety of viral strains for transfection are publicly available, e.g., the L-1 variant of Autographa californica NPV and the Bm-5 strain of Bombyx mori NPV, and such viruses may be used, particularly for transfection of Spodoptera frugiperda cells.
  • Plant cell cultures of cotton, corn, potato, soybean, petunia, tomato, and tobacco can also be utilized as hosts.
  • humanized anti-IL-23p19 is carried out in vertebrate cells.
  • the propagation of vertebrate cells in culture has become routine procedure and techniques are widely available.
  • useful mammalian host cell lines are monkey kidney CV1 line transformed by SV40 (COS-7, ATCC CRL 1651), human embryonic kidney line (293 or 293 cells subcloned for growth in suspension culture, ( Graham et al., 1977, J. Gen Virol. 36: 59 ), baby hamster kidney cells (BHK, ATCC CCL 10), Chinese hamster ovary cells/-DHFR1 (CHO, Urlaub et al., 1980, Proc. Natl. Acad. Sci.
  • Host cells are transformed with the above-described expression or cloning vectors for humanized anti-IL-23p19 antibody production and cultured in conventional nutrient media modified as appropriate for inducing promoters, selecting transformants, or amplifying the genes encoding the desired sequences.
  • the host cells used to produce a humanized anti-IL-23p19 antibody described herein may be cultured in a variety of media.
  • Commercially available media such as Ham's F10 (Sigma-Aldrich Co., St. Louis, Mo.), Minimal Essential Medium ((MEM), (Sigma-Aldrich Co.), RPMI-1640 (Sigma-Aldrich Co.), and Dulbecco's Modified Eagle's Medium ((DMEM), Sigma-Aldrich Co.) are suitable for culturing the host cells.
  • any of the media described in one or more of Ham et al., 1979, Meth. Enz. 58: 44 Barnes et al., 1980, Anal. Biochem.
  • any of these media may be supplemented as necessary with hormones and/or other growth factors (such as insulin, transferrin, or epidermal growth factor), salts (such as sodium chloride, calcium, magnesium, and phosphate), buffers (such as HEPES), nucleotides (such as adenosine and thymidine), antibiotics (such as gentamicin), trace elements (defined as inorganic compounds usually present at final concentrations in the micromolar range), and glucose or an equivalent energy source.
  • growth factors such as insulin, transferrin, or epidermal growth factor
  • salts such as sodium chloride, calcium, magnesium, and phosphate
  • buffers such as HEPES
  • nucleotides such as adenosine and thymidine
  • antibiotics such as gentamicin
  • trace elements defined as inorganic compounds usually present at final concentrations in the micromolar range
  • glucose or an equivalent energy source glucose or an equivalent energy source.
  • Other supplements may also be included at appropriate concentrations that would be
  • the antibody can be produced intracellularly, in the periplasmic space, or directly secreted into the medium. If the antibody is produced intracellularly, the cells may be disrupted to release protein as a first step. Particulate debris, either host cells or lysed fragments, can be removed, for example, by centrifugation or ultrafiltration. Carter et al., 1992, Bio/Technology 10:163-167 describes a procedure for isolating antibodies that are secreted to the periplasmic space of E. coli. Briefly, cell paste is thawed in the presence of sodium acetate (pH 3.5), EDTA, and phenylmethylsulfonylfluoride (PMSF) over about 30 minutes.
  • sodium acetate pH 3.5
  • EDTA EDTA
  • PMSF phenylmethylsulfonylfluoride
  • Cell debris can be removed by centrifugation.
  • supernatants from such expression systems are generally first concentrated using a commercially available protein concentration filter, for example, an Amicon or Millipore Pellicon ultrafiltration unit.
  • a protease inhibitor such as PMSF may be included in any of the foregoing steps to inhibit proteolysis and antibiotics may be included to prevent the growth of adventitious contaminants.
  • a variety of methods can be used to isolate the antibody from the host cell.
  • the antibody composition prepared from the cells can be purified using, for example, hydroxylapatite chromatography, gel electrophoresis, dialysis, and affinity chromatography, with affinity chromatography being a typical purification technique.
  • affinity chromatography is a typical purification technique.
  • the suitability of protein A as an affinity ligand depends on the species and isotype of any immunoglobulin Fc domain that is present in the antibody.
  • Protein A can be used to purify antibodies that are based on human gamma1, gamma2, or gamma4 heavy chains (see, e.g., Lindmark et al., 1983 J. Immunol. Meth. 62:1-13 ).
  • Protein G is recommended for all mouse isotypes and for human gamma3 (see, e.g., Guss et al., 1986 EMBO J. 5:1567-1575 ).
  • a matrix to which an affinity ligand is attached is most often agarose, but other matrices are available. Mechanically stable matrices such as controlled pore glass or poly(styrenedivinyl)benzene allow for faster flow rates and shorter processing times than can be achieved with agarose.
  • the antibody comprises a C H3 domain
  • the Bakerbond ABX TM resin J. T. Baker, Phillipsburg, N.J.
  • the mixture comprising the antibody of interest and contaminants may be subjected to low pH hydrophobic interaction chromatography using an elution buffer at a pH between about 2.5-4.5, typically performed at low salt concentrations (e.g., from about 0-0.25M salt).
  • nucleic acids that hybridize under low, moderate, and high stringency conditions, as defined herein, to all or a portion (e.g., the portion encoding the variable region) of the nucleotide sequence represented by isolated polynucleotide sequence(s) that encode an antibody of the present invention.
  • the hybridizing portion of the hybridizing nucleic acid is typically at least 15 (e.g., 20, 25, 30 or 50) nucleotides in length.
  • the hybridizing portion of the hybridizing nucleic acid is at least 80%, e.g., at least 90%, at least 95%, or at least 98%, identical to the sequence of a portion or all of a nucleic acid encoding an anti-IL-23p19 polypeptide (e.g., a heavy chain or light chain variable region), or its complement.
  • Hybridizing nucleic acids of the type described herein can be used, for example, as a cloning probe, a primer, e.g., a PCR primer, or a diagnostic probe.
  • Some reference aspects include isolated polynucleotides including sequences that encode an antibody or antibody fragment having the amino acid sequence of any one of SEQ ID NO: 84, 86, 88, 90, 91, 93, 95, 97, 99, 101, 103, 105, 107, 109, 111, 113, 115, 117 or 119, and that is at least 80%, at least 90%, at least 95%, at least 98%, or at least 99% identical to the polynucleotide sequences of SEQ ID NO:83, 85, 87, 89, 92, 94, 96, 98, 100, 102, 104, 106, 108, 110, 112, 114, 116 or 118.
  • Some reference aspects include isolated polynucleotides including sequences that encode an antibody or antibody fragment having the amino acid sequence of any one of SEQ ID NO: 158, 160, 162 or 164, and that is at least 80%, at least 90%, at least 95%, at least 98%, or at least 99% identical to the polynucleotide sequences of SEQ ID NO:157, 159, 161 or 163.
  • Some reference aspects include isolated polynucleotides including sequences that encode an antibody or antibody fragment having the amino acid sequence of any one of SEQ ID NO: 121, 123, 125, 127, 128, 130, 132, 134, 136, 138, 140, 142, 144, 146, 148, 150, 152, 154 or 156, and that is at least 80%, at least 90%, at least 95%, at least 98%, or at least 99% identical to the polynucleotide sequences of SEQ ID NO: 120, 122, 124, 126, 129, 131, 133, 135, 137, 139, 141, 143, 145, 147, 149, 151, 153 or 155.
  • Some reference aspects include isolated polynucleotides including sequences that encode an antibody or antibody fragment having the amino acid sequence of any one of SEQ ID NO: 166, 168, 170 or 172, and that is at least 80%, at least 90%, at least 95%, at least 98%, or at least 99% identical to the polynucleotide sequences of SEQ ID NO: 165, 167, 169 or 171.
  • Some reference aspects include isolated polynucleotides including sequences that encode an antibody or antibody fragment having the light chain variable region amino acid sequence that is at least 80%, at least 90%, at least 95%, at least 98%, or at least 99% identical to the amino acid sequence of any one of SEQ ID NO: 84, 86, 88, 90, 91, 93, 95, 97, 99, 101, 103, 105, 107, 109, 111, 113, 115, 117 or 119.
  • Some reference aspects include isolated polynucleotides including sequences that encode an antibody or antibody fragment having the light chain variable region amino acid sequence that is at least 80%, at least 90%, at least 95%, at least 98%, or at least 99% identical to the amino acid sequence of any one of SEQ ID NO:158, 160, 162 or 164.
  • Some reference aspects include isolated polynucleotides including sequences that encode an antibody or antibody fragment having the heavy chain variable region amino acid sequence that is at least 80%, at least 90%, at least 95%, at least 98%, or at least 99% identical to the amino acid sequence of any one of SEQ ID NO:121, 123, 125, 127, 128, 130, 132, 134, 136, 138, 140, 142, 144, 146, 148, 150, 152, 154 or 156.
  • Some reference aspects include isolated polynucleotides including sequences that encode an antibody or antibody fragment having the heavy chain variable region amino acid sequence that is at least 80%, at least 90%, at least 95%, at least 98%, or at least 99% identical to the amino acid sequence of any of one SEQ ID NO:166, 168, 170 or 172.
  • the terms "identical” or “percent identity,” in the context of two or more nucleic acids or polypeptide sequences refer to two or more sequences or subsequences that are the same or have a specified percentage of nucleotides or amino acid residues that are the same, when compared and aligned for maximum correspondence.
  • the sequences are aligned for optimal comparison purposes (e.g., gaps can be introduced in the sequence of a first amino acid or nucleic acid sequence for optimal alignment with a second amino or nucleic acid sequence).
  • the amino acid residues or nucleotides at corresponding amino acid positions or nucleotide positions are then compared. When a position in the first sequence is occupied by the same amino acid residue or nucleotide as the corresponding position in the second sequence, then the molecules are identical at that position.
  • the two sequences that are compared are the same length after gaps are introduced within the sequences, as appropriate (e.g., excluding additional sequence extending beyond the sequences being compared).
  • the leader and/or constant domain sequences are not considered.
  • a "corresponding" CDR refers to a CDR in the same location in both sequences (e.g., CDR-H1 of each sequence).
  • the determination of percent identity or percent similarity between two sequences can be accomplished using a mathematical algorithm.
  • a preferred, non-limiting example of a mathematical algorithm utilized for the comparison of two sequences is the algorithm of Karlin and Altschul, 1990, Proc. Natl. Acad. Sci. USA 87:2264-2268 , modified as in Karlin and Altschul, 1993, Proc. Natl. Acad. Sci. USA 90:5873-5877 .
  • Such an algorithm is incorporated into the NBLAST and XBLAST programs of Altschul et al., 1990, J. Mol. Biol. 215:403-410 .
  • Gapped BLAST can be utilized as described in Altschul et al., 1997, Nucleic Acids Res. 25:3389-3402 .
  • PSI-Blast can be used to perform an iterated search which detects distant relationships between molecules (Id.).
  • BLAST Gapped BLAST
  • PSI-Blast programs the default parameters of the respective programs (e.g., XBLAST and NBLAST) can be used.
  • Another preferred, non-limiting example of a mathematical algorithm utilized for the comparison of sequences is the algorithm of Myers and Miller, CABIOS (1989). Such an algorithm is incorporated into the ALIGN program (version 2.0) which is part of the GCG sequence alignment software package.
  • ALIGN program version 2.0
  • a PAM120 weight residue table, a gap length penalty of 12, and a gap penalty of 4 can be used. Additional algorithms for sequence analysis are known in the art and include ADVANCE and ADAM as described in Torellis and Robotti, 1994, Comput. Appl.
  • the antibodies described herein are useful as affinity purification agents.
  • the antibodies are immobilized on a solid phase such a Protein A resin, using methods well known in the art.
  • the immobilized antibody is contacted with a sample containing the IL-23p19 protein (or fragment thereof) to be purified, and thereafter the support is washed with a suitable solvent that will remove substantially all the material in the sample except the IL-2319 protein, which is bound to the immobilized antibody. Finally, the support is washed with another suitable solvent that will release the IL-23p19 protein from the antibody.
  • Anti-IL-23p19 antibodies are also useful in diagnostic assays to detect and/or quantify IL-23 protein, for example, detecting IL-23 expression in specific cells, tissues, or serum.
  • the anti-IL-23p19 antibodies can be used diagnostically to, for example, monitor the development or progression of a disease as part of a clinical testing procedure to, e.g., determine the efficacy of a given treatment and/or prevention regimen. Detection can be facilitated by coupling the anti-IL-23p19 antibody.
  • detectable substances include various enzymes, prosthetic groups, fluorescent materials, luminescent materials, bioluminescent materials, radioactive materials, positron emitting metals using various positron emission tomographies, and nonradioactive paramagnetic metal ions. See, for example, U.S. Patent No. 4,741,900 for metal ions which can be conjugated to antibodies for use as diagnostics.
  • the anti-IL-23p19 antibodies can be used in methods for diagnosing an IL-23-associated disorder (e.g., a disorder characterized by abnormal expression of IL-23) or to determine if a subject has an increased risk of developing an IL-23-associated disorder. Such methods include contacting a biological sample from a subject with an IL-23p19 antibody and detecting binding of the antibody to IL-23p19.
  • biological sample is intended any biological sample obtained from an individual, cell line, tissue culture, or other source of cells potentially expressing IL-23. Methods for obtaining tissue biopsies and body fluids from mammals are well known in the art.
  • the method can further comprise comparing the level of IL-23 in a patient sample to a control sample (e.g., a subject that does not have an IL-23-associated disorder) to determine if the patient has an IL-23-associated disorder or is at risk of developing an IL-23-associated disorder.
  • a control sample e.g., a subject that does not have an IL-23-associated disorder
  • the antibody can be label with a detectable moiety.
  • detectable labels are available, including radioisotopes, fluorescent labels, enzyme substrate labels and the like.
  • the label may be indirectly conjugated with the antibody using various known techniques.
  • the antibody can be conjugated with biotin and any of the three broad categories of labels mentioned above can be conjugated with avidin, or vice versa.
  • Biotin binds selectively to avidin and thus, the label can be conjugated with the antibody in this indirect manner.
  • the antibody can be conjugated with a small hapten (such as digoxin) and one of the different types of labels mentioned above is conjugated with an anti-hapten antibody (e.g., anti-digoxin antibody).
  • a small hapten such as digoxin
  • an anti-hapten antibody e.g., anti-digoxin antibody
  • radioisotopes labels include 35 S, 14 C, 125 I, 3 H, and 131 I.
  • the antibody can be labeled with the radioisotope, using the techniques described in, for example, Current Protocols in Immunology, Volumes 1 and 2, 1991, Coligen et al., Ed. Wiley-Interscience, New York, N.Y ., Pubs. Radioactivity can be measured, for example, by scintillation counting.
  • Exemplary fluorescent labels include labels derived from rare earth chelates (europium chelates) or fluorescein and its derivatives, rhodamine and its derivatives, dansyl, Lissamine, phycoerythrin, and Texas Red are available.
  • the fluorescent labels can be conjugated to the antibody via known techniques, such as those disclosed in Current Protocols in Immunology, for example. Fluorescence can be quantified using a fluorimeter.
  • the enzyme generally catalyzes a chemical alteration of the chromogenic substrate that can be measured using various techniques. For example, alteration may be a color change in a substrate that can be measured spectrophotometrically. Alternatively, the enzyme may alter the fluorescence or chemiluminescence of the substrate. Techniques for quantifying a change in fluorescence are described above.
  • the chemiluminescent substrate becomes electronically excited by a chemical reaction and may then emit light that can be measured, using a chemiluminometer, for example, or donates energy to a fluorescent acceptor.
  • enzymatic labels include luciferases such as firefly luciferase and bacterial luciferase ( U.S. Pat. No. 4,737,456 ), luciferin, 2,3-dihydrophthalazinediones, malate dehydrogenase, urease, peroxidase such as horseradish peroxidase (HRPO), alkaline phosphatase, ⁇ -galactosidase, glucoamylase, lysozyme, saccharide oxidases (such as glucose oxidase, galactose oxidase, and glucose-6-phosphate dehydrogenase), heterocydic oxidases (such as uricase and xanthine oxidase), lactoperoxidase, microperoxidase, and the like.
  • luciferases such as firefly luciferase and bacterial luciferase ( U.S. Pat.
  • enzyme-substrate combinations include, for example: Horseradish peroxidase (HRPO) with hydrogen peroxidase as a substrate, wherein the hydrogen peroxidase oxidizes a dye precursor such as orthophenylene diamine (OPD) or 3,3',5,5'-tetramethyl benzidine hydrochloride (TMB); alkaline phosphatase (AP) with para-Nitrophenyl phosphate as chromogenic substrate; and ⁇ -D-galactosidase ( ⁇ -D-Gal) with a chromogenic substrate such as p-nitrophenyl- ⁇ -D-galactosidase or fluorogenic substrate 4-methylumbelliferyl- ⁇ -D-galactosidase.
  • HRPO Horseradish peroxidase
  • OPD orthophenylene diamine
  • TMB 3,3',5,5'-tetramethyl benzidine hydrochloride
  • AP alkaline phosphatase
  • the humanized anti-IL-23p19 antibody is used unlabeled and detected with a labeled antibody that binds the humanized anti-IL-23p19 antibody.
  • the antibodies described herein may be employed in any known assay method, such as competitive binding assays, direct and indirect sandwich assays, and immunoprecipitation assays. See, e.g., Zola, Monoclonal Antibodies: A Manual of Techniques, pp. 147-158 (CRC Press, Inc. 1987 ).
  • the anti-IL-23p19 antibody or antigen binding fragment thereof can be used to inhibit the binding of IL-23 to the IL-23 receptor.
  • Such methods comprise administering an anti-IL-23p19 antibody or antigen binding fragment thereof to a cell (e.g., a mammalian cell) or cellular environment, whereby signaling mediated by the IL-23 receptor is inhibited. These methods can be performed in vitro or in vivo.
  • cellular environment is intended the tissue, medium, or extracellular matrix surrounding a cell.
  • the anti-IL-23p19 antibody or antigen binding fragment thereof is administered to the cellular environment of a cell in such a manner that the antibody or fragment is capable of binding to IL-23 molecules outside of and surrounding the cell, therefore, preventing the binding of IL-23 to its receptor.
  • An anti-IL-23p19 antibody can be used in a diagnostic kit, i.e., a packaged combination of reagents in predetermined amounts with instructions for performing the diagnostic assay.
  • the kit may include substrates and cofactors required by the enzyme such as a substrate precursor that provides the detectable chromophore or fluorophore.
  • other additives may be included such as stabilizers, buffers (for example a block buffer or lysis buffer), and the like.
  • the relative amounts of the various reagents may be varied widely to provide for concentrations in solution of the reagents that substantially optimize the sensitivity of the assay.
  • the reagents may be provided as dry powders, usually lyophilized, including excipients that on dissolution will provide a reagent solution having the appropriate concentration.
  • a humanized anti-IL-23p19 antibody disclosed herein is useful in the treatment of various disorders associated with the expression of IL-23p19 as described herein.
  • Methods for treating an IL-23 associated disorder comprise administering a therapeutically effective amount of a humanized anti-IL-23p19 antibody to a subject in need thereof.
  • the humanized anti-IL-23p19 antibody or agent is administered by any suitable means, including parenteral, subcutaneous, intraperitoneal, intrapulmonary, and intranasal, and, if desired for local immunosuppressive treatment, intralesional administration (including perfusing or otherwise contacting the graft with the antibody before transplantation).
  • the humanized anti-IL-23p19 antibody or agent can be administered, for example, as an infusion or as a bolus.
  • Parenteral infusions include intramuscular, intravenous, intraarterial, intraperitoneal, or subcutaneous administration.
  • the humanized anti-IL-23p19 antibody is suitably administered by pulse infusion, particularly with declining doses of the antibody.
  • the dosing is given by injections, most preferably intravenous or subcutaneous injections, depending in part on whether the administration is brief or chronic.
  • the appropriate dosage of antibody will depend on a variety of factors such as the type of disease to be treated, as defined above, the severity and course of the disease, whether the antibody is administered for preventive or therapeutic purposes, previous therapy, the patient's clinical history and response to the antibody, and the discretion of the attending physician.
  • the antibody is suitably administered to the patient at one time or over a series of treatments.
  • ⁇ g/kg to 20 mg/kg (e.g., 0.1-15 mg/kg) of antibody is an initial candidate dosage for administration to the patient, whether, for example, by one or more separate administrations, or by continuous infusion.
  • a typical daily dosage might range from about 1 ⁇ g/kg to 100 mg/kg or more, depending on the factors mentioned above.
  • the treatment is sustained until a desired suppression of disease symptoms occurs.
  • other dosage regimens may be useful. The progress of this therapy is easily monitored by conventional techniques and assays.
  • An exemplary dosing regimen is that disclosed in WO 94/04188 .
  • suppression is used herein in the same context as “amelioration” and “alleviation” to mean a lessening of one or more characteristics of the disease.
  • the antibody composition will be formulated, dosed, and administered in a fashion consistent with good medical practice.
  • Factors for consideration in this context include the particular disorder being treated, the particular mammal being treated, the clinical condition of the individual patient, the cause of the disorder, the site of delivery of the agent, the method of administration, the scheduling of administration, and other factors known to medical practitioners.
  • the "therapeutically effective amount" of the antibody to be administered will be governed by such considerations, and is the minimum amount necessary to prevent, ameliorate, or treat the disorder associated with IL-23 expression.
  • the antibody need not be, but is optionally, formulated with one or more agents currently used to prevent or treat the disorder in question.
  • the effective amount of such other agents depends on the amount of humanized anti-IL-23p19 antibody present in the formulation, the type of disorder or treatment, and other factors discussed above. These are generally used in the same dosages and with administration routes as used hereinbefore or about from 1 to 99% of the heretofore employed dosages.
  • the anti-IL-23p19 antibodies or agents are useful for treating or preventing an immunological disorder characterized by abnormal expression of IL-23, e.g., by inappropriate activation of immune cells (e.g., lymphocytes or dendritic cells). Such abnormal expression of IL-23 can be due to, for example, increased IL-23 protein levels.
  • the anti-IL-23p19 antibodies or antigen binding fragments thereof also find use in the treatment or prevention of respiratory disorders, metabolic disorders, for example diabetes mellitus, and certain cancers.
  • Treatment or prevention of the immunological disorder, respiratory disorder, metabolic disorder or cancer, according to the methods described herein, is achieved by administering to a subject in need of such treatment or prevention an effective amount of the anti-IL-23p19 antibody or agent, whereby the antibody decreases the activity of IL-23 associated with the disease state.
  • Immunological diseases that are characterized by inappropriate activation of immune cells and that can be treated or prevented by the methods described herein can be classified, for example, by the type(s) of hypersensitivity reaction(s) that underlie the disorder. These reactions are typically classified into four types: anaphylactic reactions, cytotoxic (cytolytic) reactions, immune complex reactions, or cell-mediated immunity (CMI) reactions (also referred to as delayed-type hypersensitivity (DTH) reactions).
  • CMI cell-mediated immunity
  • DTH delayed-type hypersensitivity
  • Immunological diseases include inflammatory diseases and autoimmune diseases.
  • immunological diseases include the following: rheumatoid arthritis, autoimmune demyelinative diseases (e.g., multiple sclerosis, allergic encephalomyelitis), endocrine opthalmopathy, uveoretinitis, systemic lupus erythematosus, myasthenia gravis, Grave's disease, glomerulonephritis, autoimmune hepatological disorder, inflammatory bowel disease (e.g., Crohn's disease or ulcerative colitis), anaphylaxis, allergic reaction, Sjogren's syndrome, type I diabetes mellitus, primary biliary cirrhosis, Wegener's granulomatosis, fibromyalgia, polymyositis, dermatomyositis, inflammatory myositis, multiple endocrine failure, Schmidt's syndrome, autoimmune uveitis, Addison's disease, adrenalitis, thyroiditis, Hashimoto's thyroiditis,
  • the immunological disease is psoriasis.
  • Psoriasis is a chronic inflammatory disease of the skin characterized by dysfunctional keratinocyte differentiation and hyperproliferation and marked accumulation of inflammatory T cells and dendritic cells.
  • the immunological disease includes plaque psoriasis, for example chronic plaque psoriasis, for example moderate to severe chronic plaque psoriasis, for example in patients who are candidates for systemic therapy or phototherapy.
  • the immunological disease includes palmar pustular psoriasis, guttate psoriasis, inverse psoriasis, pustular psoriasis or erythodermic psoriasis.
  • the immunological disease is Crohn's disease.
  • Crohn's disease is an inflammatory bowel disease characterized by chronic transmural inflammatory lesions in the gastrointestinal epithelial barrier and mucosa. CD can affect any site within the GI tract, most frequently the terminal ileum. CD typically follows a relapsing and remitting course, and causes substantial acute and long-term morbidity and increased mortality. Patients often develop local complications (e.g. fistulas, abscesses), systemic complications (e.g. uveitis, arthritis), or side effects of treatment, and may require major surgery. Roughly a third of patients fall into each of the categories of mild, moderate, and severe disease. Patients with moderate-to-severe Crohn's Disease have debilitating illness with clinical features such as abdominal pain, diarrhea, abscess formation, and fistulae.
  • the immunological disease is spondyloarthritis.
  • the disease is ankylosing spondylitis (also called radiographic spondyloarthritis).
  • Ankylosing spondylitis (AS; radiographic axial spondyloarthritis) is the prototypical form of the spondyloarthritides (SpA). It is a crippling insidious rheumatologic disease that is commoner in males than females and affects young individuals; in 80% of patients the onset of symptoms occurs at less than 30 years of age.
  • an inflammatory component which contributes to the Ankylosing Spondylitis Activity Score (ASAS) that is used as a measure of improvement in treatment studies.
  • ASS Ankylosing Spondylitis Activity Score
  • abnormal bone growth results in the formation of bony spurs (syndesmophytes) visible on plain radiographs accompanied by increased spinal rigidity, ultimately leading to bony ankylosis and vertebral deformities.
  • the immunological disease is non-radiographic axial Spondyloarthritis.
  • Non-radiographic axial Spondyloarthritis a more recently defined entity than AS, is considered to represent an earlier manifestation of the same pathologic processes as AS, although it is increasingly recognized that some patients (particularly females) may not progress to radiographic disease, and may therefore be considered to have a distinct subtype of this disease.
  • the disease is peripheral spondyloarthritis.
  • Peripheral spondyloarthritis can be defined by the presence of arthritis, enthesitis, or dactylitis, with either at least one 'specific' SpA feature (psoriasis, inflammatory bowel disease, preceding infection, HLA-B27, uveitis, sacroiliitis on imaging) or at least two of the remaining SpA-features (arthritis, enthesitis, dactylitis, inflammatory back pain in the past, positive family history for SpA).
  • This definition is for example used by ASAS (Association SpondyloArthritis investigation Society), see also for example Carron P. et al. (Curr Opinion Rheumatol 2012; 24: 370-4 ).
  • the immunological disorder is a T cell-mediated immunological disorder and accordingly, the anti-IL-23p19 antibodies and agents as described herein are also useful for treating or preventing T cell-mediated immunological disorders.
  • the anti-IL-23p19 antibodies or agents are useful for treating or preventing a respiratory disorder in which IL-23 is abnormally expressed.
  • Treatment or prevention of the respiratory disorder is achieved by administering to a subject in need of such treatment or prevention an effective amount of the anti-IL-23p19 antibody or agent, whereby the antibody decreases the activity of IL-23 associated with the disease state.
  • respiratory complaints include, but are not limited to: respiratory complaints, obstructive pulmonary diseases of various origins, pulmonary emphysema of various origins, restrictive pulmonary diseases, interstitial pulmonary diseases, interstitial lung disease, cystic fibrosis, bronchitis of various origins, bronchiectasis, ARDS (adult respiratory distress syndrome) and all forms of pulmonary oedema; obstructive pulmonary diseases selected from among COPD (chronic obstructive pulmonary disease), asthma, bronchial asthma, paediatric asthma, severe asthma, acute asthma attacks and chronic
  • the anti-IL-23p19 antibodies and agents as described herein are also useful for treating cancers, in which IL-23 is abnormally expressed.
  • IL-23-expressing cancers that can be treated by the methods described herein include, for example, leukemia, such as acute leukemia, acute lymphocytic leukemia, acute myelocytic leukemia (e.g., myeloblastic, promyelocytic, myelomonocytic, monocytic, or erythroleukemia), chronic leukemia, chronic myelocytic (granulocytic) leukemia, or chronic lymphocytic leukemia; Polycythemia vera; Lymphoma (e.g., Hodgkin's disease or Non-Hodgkin's disease); multiple myeloma, Waldenstrom's macroglobulinemia; heavy chain disease; solid tumors such sarcomas and carcinomas (e.g., fibrosarcoma, myxosarcoma, liposarcoma, chondrosarcoma, osteogenic sarcoma, osteosarcoma, chordoma
  • the anti-IL-23p19 antibodies and agents as described herein are also useful for treating discoid lupus, malaria, malignant melanoma, aplastic anaemia, Huntington's Disease, palmoplantar pustulosis, IgA nephritis, ANCA-Associated Vasculitis, scleritis or sepsis.
  • a composition comprising the anti-IL-23p19 antibody as claimed can be administered to a subject having or at risk of having an immunological disorder, respiratory disorder or a cancer.
  • the invention further provides for the use of the anti-IL-23p19 antibody in the manufacture of a medicament for prevention or treatment of a cancer, respiratory disorder or immunological disorder.
  • subject as used herein means any mammalian patient to which an IL-23p19 binding agent can be administered, including, e.g., humans and non-human mammals, such as primates, rodents, and dogs. Subjects specifically intended for treatment using the methods described herein include humans.
  • the antibodies can be administered either alone or in combination with other compositions in the prevention or treatment of the immunological disorder, respiratory disorder or cancer.
  • Such compositions which can be administered in combination with the antibodies or agents include methotrexate (MTX) and immunomodulators, e.g. antibodies or small molecules.
  • MTX methotrexate
  • immunomodulators e.g. antibodies or small molecules.
  • IL-23p19 binding agent can be administered, for example by infusion, bolus or injection, and can be administered together with other biologically active agents such as chemotherapeutic agents. Administration can be systemic or local. In preferred embodiments, the administration is by subcutaneous injection. Formulations for such injections may be prepared in for example prefilled syringes that may be administered once every other week.
  • the IL-23p19 antibody composition is administered bv injection, by means of a catheter, by means of a suppository, or by means of an implant, the implant being of a porous, non-porous, or gelatinous material, including a membrane, such as a sialastic membrane, or a fiber.
  • a membrane such as a sialastic membrane, or a fiber.
  • the anti-IL-23p19 antibody composition is delivered in a controlled release system.
  • a pump may be used (see, e.g., Langer, 1990, Science 249:1527-1533 ; Sefton, 1989, CRC Crit. Ref. Biomed. Eng. 14:201 ; Buchwald et al., 1980, Surgery 88:507 ; Saudek et al., 1989, N. Engl. J. Med. 321:574 ).
  • polymeric materials can be used.
  • An IL-23p19 anti-IL-23p19 antibody can be administered as pharmaceutical compositions comprising a therapeutically effective amount of the binding agent and one or more pharmaceutically compatible ingredients.
  • the pharmaceutical composition is formulated in accordance with routine procedures as a pharmaceutical composition adapted for intravenous or subcutaneous administration to human beings.
  • compositions for administration by injection are solutions in sterile isotonic aqueous buffer.
  • the pharmaceutical can also include a solubilizing agent and a local anesthetic such as lignocaine to ease pain at the site of the injection.
  • the ingredients are supplied either separately or mixed together in unit dosage form, for example, as a dry lyophilized powder or water free concentrate in a hermetically sealed container such as an ampoule or sachette indicating the quantity of active agent.
  • the pharmaceutical is to be administered by infusion, it can be dispensed with an infusion bottle containing sterile pharmaceutical grade water or saline.
  • an ampoule of sterile water for injection or saline can be provided so that the ingredients can be mixed prior to administration.
  • the pharmaceutical composition can be provided as a pharmaceutical kit comprising (a) a container containing a IL-23p19 , an anti-IL-23p19 antibody in lyophilized form and (b) a second container containing a pharmaceutically acceptable diluent (e.g., sterile water) for injection.
  • a pharmaceutically acceptable diluent e.g., sterile water
  • the pharmaceutically acceptable diluent can be used for reconstitution or dilution of the lyophilized anti-IL-23p19 antibody or agent.
  • Optionally associated with such container(s) can be a notice in the form prescribed by a governmental agency regulating the manufacture, use or sale of pharmaceuticals or biological products, which notice reflects approval by the agency of manufacture, use or sale for human administration.
  • the amount of the IL-23p19 binding agent anti-IL-23p19 antibody that is effective in the treatment or prevention of an immunological disorder or cancer can be determined by standard clinical techniques.
  • in vitro assays may optionally be employed to help identify optimal dosage ranges.
  • the precise dose to be employed in the formulation will also depend on the route of administration, and the stage of immunological disorder or cancer, and should be decided according to the judgment of the practitioner and each patient's circumstances. Effective doses may be extrapolated from dose-response curves derived from in vitro or animal model test systems.
  • the dosage of an anti-IL-23p19 antibody administered to a patient with an immunological disorder or IL-23p19-expressing cancer is typically about 0.1 mg/kg to about 100 mg/kg of the subject's body weight.
  • the dosage administered to a subject is about 0.1 mg/kg to about 50 mg/kg, about 1 mg/kg to about 30 mg/kg, about 1 mg/kg to about 20 mg/kg, about 1 mg/kg to about 15 mg/kg, or about 1 mg/kg to about 10 mg/kg of the subject's body weight.
  • Exemplary doses include, but are not limited to, from 1 ng/kg to 100 mg/kg.
  • a dose is about 0.5 mg/kg, about 1 mg/kg, about 2 mg/kg, about 3 mg/kg, about 4 mg/kg, about 5 mg/kg, about 6 mg/kg, about 7 mg/kg, about 8 mg/kg, about 9 mg/kg, about 10 mg/kg, about 11 mg/kg, about 12 mg/kg, about 13 mg/kg, about 14 mg/kg, about 15 mg/kg or about 16 mg/kg.
  • the dose can be administered, for example, daily, once per week (weekly), twice per week, thrice per week, four times per week, five times per week, six times per week, biweekly or monthly, every two months, or every three months.
  • the dose is about 0.5 mg/kg/week, about 1 mg/kg/week, about 2 mg/kg/week, about 3 mg/kg/week, about 4 mg/kg/week, about 5 mg/kg/week, about 6 mg/kg/week, about 7 mg/kg/week, about 8 mg/kg/week, about 9 mg/kg/week, about 10 mg/kg/week, about 11 mg/kg/week, about 12 mg/kg/week, about 13 mg/kg/week, about 14 mg/kg/week, about 15 mg/kg/week or about 16 mg/kg/week.
  • the dose ranges from about 1 mg/kg/week to about 15 mg/kg/week.
  • the pharmaceutical compositions comprising the IL-23p19 binding agent can further comprise a therapeutic agent, either conjugated or unconjugated to the binding agent.
  • the anti-IL-23p19 antibody can be co-administered in combination with one or more therapeutic agents for the treatment or prevention of immunological disorders or cancers.
  • Such combination therapy administration can have an additive or synergistic effect on disease parameters (e.g., severity of a symptom, the number of symptoms, or frequency of relapse).
  • disease parameters e.g., severity of a symptom, the number of symptoms, or frequency of relapse.
  • an anti-IL-23p19 antibody is administered concurrently with a therapeutic agent.
  • the therapeutic agent is administered prior or subsequent to administration of the anti-IL-23p19 antibody , by at least an hour and up to several months, for example at least an hour, five hours, 12 hours, a day, a week, a month, or three months, prior or subsequent to administration of the anti-IL-23p19 antibody or IL-23p19 binding agent.
  • a pharmaceutical composition of the present invention comprises an antibody molecule, namely the antibody molecule as claimed and further comprises 90 mg/ml of said antibody and 0.16 mmol/l Polysorbate 20, wherein said pharmaceutical composition further comprises (i) 240 mmol/l sorbitol, or (ii) 0.5 mmol/l Succinic acid, 3.9 mmol/l Disodium succinate hexahydrate, and 225 mmol/l Sorbitol, wherein the pH of said pharmaceutical composition is in the range of pH 5.5 to 6.5.
  • compositions according to the present invention are described in further details hereinbelow.
  • the pharmaceutical compositions disclosed herein are physico-chemically stable and maintain the Integrity of an antibody comprised in said pharmaceutical composition, for example when stored for 8 weeks at 40°C as shown hereinbelow.
  • an article of manufacture containing materials useful for the treatment of the disorders described above comprises a container and a label.
  • Suitable containers include, for example, bottles, vials, syringes, and test tubes.
  • the containers may be formed from a variety of materials such as glass or plastic.
  • the container holds a composition that is effective for treating the condition and may have a sterile access port.
  • the container may be an intravenous solution bag or a vial having a stopper pierceable by a hypodermic injection needle.
  • the active agent in the composition is the humanized anti-IL-23p19 antibody.
  • the label on or associated with the container indicates that the composition is used for treating the condition of choice.
  • the article of manufacture may further comprise a second container comprising a pharmaceutically-acceptable buffer, such as phosphate-buffered saline, Ringer's solution, and dextrose solution. It may further include other materials desirable from a commercial and user standpoint, including other buffers, diluents, filters, needles, syringes, and package inserts with instructions for use.
  • a pharmaceutically-acceptable buffer such as phosphate-buffered saline, Ringer's solution, and dextrose solution.
  • It may further include other materials desirable from a commercial and user standpoint, including other buffers, diluents, filters, needles, syringes, and package inserts with instructions for use.
  • Mouse lead antibody 6B8 was converted to a chimeric antibody consisting of the mouse variable domain of 6B8 and a human constant IgG1KO domain.
  • Mouse antibody 6B8 is shown in Tables 1 and 2 herein above.
  • the IgG1KO ( k nock ⁇ o ut) has two replacement mutations (Leu234Ala and Leu235Ala) that eliminate ADCC and CDC activity by reducing effector functions such as FcyR and complement binding.
  • the variable domains of the mouse and chimeric antibodies are identical. Chimeric antibodies are generated to confirm the function of the antibody and to ensure the correct sequence has been obtained.
  • the variable region of the antibody is then humanized through a design and screening process.
  • a library was made where human and mouse residues were varied in such a way that in any given position there could be either a human or mouse residue. Such a library was made for those amino acids that were different between human germline and mouse antibody. Only the clones that retain the function of the parent mouse antibody were selected. Representative humanized variable regions for antibody 6B8 are shown in Tables 5 and 6.
  • Antibodies A, B, C and D are shown in Table 7.
  • Antibody 6H12 (disclosed in WO 2007/027714 ), antibody QF20 (disclosed in WO 2007/024846 ) and antibody C1273 (disclosed in WO 2007/005955 ) were also tested.
  • Table 11 Antibody K D (pM) k a (1/Ms) k d (1/s) Antibody A ⁇ 1 2.95E+06 ⁇ 1e-6 Antibody B ⁇ 1 2.99E+06 ⁇ 1e-6 Antibody C 2.9 3.23E+06 9.36E-06 Antibody D 15.9 2.07E+06 3.29E-05 C-1273 >5,000 n/a n/a 6H12 157 9.91E+05 1.56E-04 QF20 1.2 3.90E+06 4.78E-06 D) Molecular Selectivity over human IL-12 The anti-IL-23p19 antibodies were also injected over a human IL-12 surface at a concentration of 100 nM.
  • the binding signal for these antibodies measured using the Fortebio Octet is zero, which indicates that these antibodies selectively bind to human IL-23.
  • the binding of the anti-IL-23p19 antibodies to IL-23 was also analyzed in the presence of 50% human serum and no significant effect of serum on binding on-rate was observed demonstrating high specificity.
  • Example 3 Competition binding assay of human IL-23 binding to human IL-23R/Fc
  • Human IL-23R-Fc was captured on the biosensor surface and 10 nM of human IL-23 was injected. The sensorgram indicates the specific binding between IL-23 and the IL-23 receptor ( Figure 2 , top trace). Antibodies were then co-injected with 10 nM human IL-23 to assess whether antibody binding to the IL-23 could inhibit the interaction between IL-23 and the IL-23 receptor. In this example, if the antibody binds to human IL-23 and is able to inhbit the interaction then reduced or no binding will be observed ( Figure 2 , bottom trace). In the example shown an equivalent molar concentration of Antibody A was co-injected with 10 nM recombinant human IL-23.
  • Example 4 Functional Cell Assays, Inhibition of IL-17 production from IL-23 stimulated mouse splenocytes
  • One functional cell assay for anti-IL-23p19 antibodies measures their ability to inhibit IL-23 stimulated IL-17 production from mononuclear cells isolated from mouse spleens.
  • Human recombinant IL-23 protein is capable of stimulating IL-17 release from mouse splenocytes.
  • a natural source of human IL-23 found in the supernatant of activated human monocytic THP-1 cells can be used to stimulate IL-17 production from mouse mononuclear cells.
  • Human recombinant IL-23 or natural human IL-23 from activated THP-1 cells was preincubated with titrated anti-IL-23p19 antibodies.
  • the IL-23/antibody combinations were then added to freshly isolated murine splenocytes.
  • Recombinant IL-23 alone was used as a positive control.
  • cell supernatants were collected and assayed for IL-17 by ELISA (R&D Systems, Minneapolis, MN). Representative IC 50 values for anti-IL-23p19 antibodies are shown below.
  • the tested antibodies are mouse antibodies derived from hybridomas (rows 1-19, see tables 1 and 2), chimeric antibodies (rows 20-23), and Antibodies A to D.
  • Antibody 6H12 (disclosed in WO 2007/027714 ), antibody QF20 (disclosed in WO 2007/024846 ) and antibody C1273 (disclosed in WO 2007/005955 ) were also tested.
  • Table 12 Antibody IC 50 Values (pM), recombinant human IL-23 IC 50 Values (pM), natural human IL-23 18C4 471 100,413 18E5 not determined 9, 9, 13 18D3 234 not determined 20E8 not determined 438, 561 22E2 61, 130 117,35 24A5 22, 37 85, 31 15C11 126 232 43F5 250, 8000 8000 27G8 235 5000 31H9 960 2000 2D1 not determined 2336, 1911, 1597 9D12 59 281, 138 6B8 13 8,2 73H10 1411 not determined 74H3 1352 not determined 36H8 not determined not determined 26F7 27 2,8 34G3 336 27, 25 34D9 510 456 Chimeric
  • Example 5 Functional specificity testing against IL-12 in a human activated T cell assay
  • Anti-IL-23p19 antibodies were tested for functional inhibition of IL-12 in a human activated T cell assay.
  • Human recombinant IL-12 (1 ng/ml) was preincubated with 5 ⁇ g/ml anti-IL-23p19 antibodies. The IL-12/antibody combinations were then added to human PHA-derived T cell blasts. Recombinant IL-12 alone was used as a positive control.
  • An anti-IL-12p70 antibody (Bender MedSystems, Vienna, Austria) was used as a control inhibitory antibody. After two days in culture, cell supernatants were collected and assayed for IFN-y by ELISA (R&D Systems). Samples were tested in triplicate and the average pg/ml of IFN- ⁇ was determined.
  • Example 6 Inhibition of IL-23 induced STAT3 phosphorylation in the human cell line DB
  • the human cell line DB (ATCC, Manassas, VA) responds to IL-23 stimulation through an endogenous IL-23Rcomplex (IL-23R and IL-12R ⁇ 1) and phosphorylates STAT3 in an IL-23 dose dependent manner.
  • An assay was developed for testing anti-IL-23p19 antibody inhibition of IL-23 induced STAT3 phosphorylation.
  • DB cells were plated at 1 ⁇ 10e6 cells/well in a 96 well plate.
  • Antibodies to be tested were serially diluted and preincubated with recombinant human IL-23 (10 ng/ml) for 1 hour at room temperature. The antibody/IL-23 mixture was then added to the cells for 30 minutes at 37°C.
  • Antibody IC 50 values were calculated as percent inhibition of STAT3 phosphorylation compared to control wells without antibody. Representative IC 50 values are shown in the table below. Table 14 Antibody IC 50 (pM) Antibody A 25, 15, 38, 23, 13, 18 Antibody B 73, 84 Antibody C 132, 80 Antibody D 158 QF20 26, 26, 27 C-1273 163,438
  • Example 7 In vivo model of IL-23 induced cytokine production in the mouse ear
  • Humanized anti-IL-23p19 antibodies were administered by ten minute intravenous infusion at a dose of 1.0 mg/kg to three cynomolgus monkeys. Serum samples were collected over a 6 week time course and free antibody concentrations were measured using a specific ELISA. The serum concentration-time profiles for the antibodies and the corresponding pharmacokinetic parameters are summarized in the Table 16 below.
  • NS0 cells were grown in the presence of 1% FBS before transfection. 40 ⁇ 10e6 cells were collected and resuspended in 0.8ml in media containing 2% FBS with 20ug of linearized DNA (heavy chain and light chain expression vectors) and then cells were incubated on ice for approximately 15 min before electroporation of the cells at 750V/25uF (Bio-Rad Gene Pulser Xcell). Cells were recovered with 2% FBS for approximately 48 hours at 37°C and 5% CO2 then plated in 96 well plates at 2 ⁇ 10e5 cells/ml containing G418 and mycophenolic acid for 14-21 days until formation of colonies.
  • Proteins were resuspended in a final buffer containing 20mM Sodium Citrate and 115mM NaCl, pH 6.0 and are stable at 4 °C for at least 4 months and with solubility up to 100 mg/ml in this buffer.
  • AUC Analytical Ultracentrifugation as measured by the sedimentation velocity method at concentrations of 0.5 -1 mg/ml; SEC: Size exclusion chromatography; %M: percent monomer.
  • Hydrogen/Deuterium Exchange Mass Spectrometry was employed to map the epitope of Antibody A binding to human IL-23p19. This method determined the susceptibility of the amide backbone hydrogens of IL-23p19 to exchange with D 2 O. The experiment was conducted with IL-23 alone and IL-23 with added Antibody A. Regions of the IL-23p19 sequence showing significant protection from exchange due to binding of Antibody A were thus identified. Resolution of the method is determined by the peptides produced by digestion with pepsin or Protease XVIII. These IL-23p19 derived peptides were identified by additional control experiments with unexchanged samples employing standard accurate mass and HPLC MS/MS technologies.
  • IL-23 Recombinant human IL-23 was used.
  • 50ul of IL-23 (0.8mg/ml) was incubated with 10ul of Antibody A (12.7mg/ml) for 15 minutes at room temperature.
  • the final molar ratio Antibody A/IL-23 was 1.2:1.
  • Pepsin and Protease XVIII peptides were identified using fragmentation data and the program Proteome Discoverer (Thermoscientific, Waltham, MA). Identified peptides were visually compared (protein alone vs. protein with antibody present) using Xcalibur software (Thermoscientific). No significant shifts in exchange were observed for IL-23 alone vs. IL-23 with Antibody A outside of the IL-23p19 region. For the p19 portion of the protein, data was analyzed using the program PepMap (Thermoscientific). This program calculates the average mass for exchanged peptides. PepMap results were checked and those peptides that did not yield verified results were calculated with the aid of Microsoft Excel.
  • the regions of the IL-23 sequence showing significant protection from exchange due to binding of Antibody A were identified as amino acid residues 108 to 126 of SEQ ID NO:181 and amino acid residues 137 to 151 of SEQ ID NO:181.
  • the pH of formulation 1 is typically in the range of pH 6.0 to 7.0, for example pH 6.5. This formulation is particularly suitable for intravenous administration.
  • the osmolarity of the formulation is 300 +/- 30 mOsmol/kg, as determined using an Osmomat 030 (Gonotec GmbH, Berlin, Germany).
  • the density at 20°C of the formulation is approximately 1.0089 g/cm 3 , as determined using a measuring unit DMA 4500 (Anton Paar GmbH, Ostfildern-Scharnhausen, Germany).
  • the pH of formulation 2 is typically in the range of pH 5.5 to 6.5, for example 5.5 to 6.1, for example the pH is 5.8.
  • This formulation is particularly suitable for subcutaneous administration.
  • the osmolarity of the formulation is 300 +/- 30 mOsmol/kg, as determined using an Osmomat 030 (Gonotec GmbH, Berlin, Germany).
  • the density at 20°C of the formulation is approximately 1.040 g/cm 3 , as determined using a measuring unit DMA 4500 (Anton Paar GmbH, Ostfildern-Scharnhausen, Germany).
  • the pH of formulation 3 is typically in the range of pH 5.5 to 6.5, for example 5.5 to 6.1, for example the pH is 5.8.
  • This formulation is particularly suitable for subcutaneous administration.
  • the osmolarity of the formulation is 300 +/- 30 mOsmol/kg, as determined using an Osmomat 030 (Gonotec GmbH, Berlin, Germany).
  • compositions are stored at 40°C for 8 weeks in a syringe in the case of formulations 2 and 3. Properties of the formulations are measured initially and after 8 weeks in storage at 40°C and are shown below.
  • the turbidity is expressed in Formazin Nephelometric Unit (FNU), as measured using nephelometry.
  • the % monomer is determined by High-Performance Size-Exclusion Chromatography (HP-SEC).
  • HP-SEC High-Performance Size-Exclusion Chromatography

Claims (11)

  1. Pharmazeutische Zusammensetzung, umfassend einen Anti-IL-23p19-Antikörper, wobei der Antikörper umfasst
    eine leichte Kette, umfassend die Aminosäuresequenz von SEQ ID NO: 174, und eine schwere Kette, umfassend die Aminosäuresequenz von SEQ ID NO: 176, wobei die pharmazeutische Zusammensetzung umfasst:
    90 mg/ml des Antikörpers und 0,16 mmol/l Polysorbat 20, wobei die pharmazeutische Zusammensetzung weiterhin umfasst: (i) 240 mmol/l Sorbit oder (ii) 0,5 mmol/l Bernsteinsäure, 3,9 mmol/l Dinatriumsuccinathexahydrat und 225 mmol/l Sorbit, wobei der pH-Wert der pharmazeutischen Zusammensetzung im Bereich von pH 5,5 bis 6,5 liegt.
  2. Pharmazeutische Zusammensetzung gemäß Anspruch 1 zur Verwendung bei der Behandlung eines Subjekts mit einer mit IL-23 assoziierten Störung, wobei der Antikörper an humanes IL-23 bindet, wobei die Erkrankung eine inflammatorische Erkrankung, eine Autoimmunerkrankung, eine Atemwegserkrankung, eine Stoffwechselstörung oder Krebs ist.
  3. Pharmazeutische Zusammensetzung zur Verwendung gemäß Anspruch 2, wobei die Erkrankung Psoriasis, entzündliche Darmerkrankung, Psoriasis-Arthritis, Multiple Sklerose, rheumatoide Arthritis oder Spondyloarthritis ist.
  4. Pharmazeutische Zusammensetzung zur Verwendung gemäß Anspruch 2, wobei die Erkrankung Plaque-Psoriasis, chronische Plaque-Psoriasis oder moderate bis schwere chronische Plaque-Psoriasis ist.
  5. Pharmazeutische Zusammensetzung zur Verwendung gemäß Anspruch 2, wobei die Erkrankung palmare pustulöse Psoriasis, Psoriasis guttata, Psoriasis inversa, pustulöse Psoriasis oder erythrodermische Psoriasis ist.
  6. Pharmazeutische Zusammensetzung zur Verwendung gemäß Anspruch 2, wobei die Erkrankung Morbus Crohn oder ulzerative Colitis ist.
  7. Pharmazeutische Zusammensetzung zur Verwendung gemäß Anspruch 2, wobei die Erkrankung ankylosierende Spondylitis, nicht-radiologische axiale Spondyloarthritis oder periphere Spondyloarthritis ist.
  8. Pharmazeutische Zusammensetzung zur Verwendung gemäß Anspruch 2, wobei die Erkrankung Lupus discoideus, Malaria, malignes Melanom, aplastische Anämie, Chorea Huntington, palmoplantare Pustulose, IgA-Nephritis, ANCA-assoziierte Vaskulitis, Skleritis oder Sepsis ist.
  9. Pharmazeutische Zusammensetzung gemäß Anspruch 1, wobei die pharmazeutische Zusammensetzung umfasst:
    90 mg/ml des Antikörpers und 0,16 mmol/l Polysorbat 20, und wobei die pharmazeutische Zusammensetzung weiterhin 240 mmol/l Sorbit umfasst.
  10. Pharmazeutische Zusammensetzung gemäß Anspruch 1, wobei die pharmazeutische Zusammensetzung umfasst:
    90 mg/ml des Antikörpers und 0,16 mmol/l Polysorbat 20, und wobei die pharmazeutische Zusammensetzung weiterhin 0,5 mmol/l Bernsteinsäure, 3,9 mmol/l Dinatriumsuccinathexahydrat und 225 mmol/l Sorbit umfasst.
  11. Pharmazeutische Zusammensetzung gemäß Anspruch 1, wobei die pharmazeutische Zusammensetzung im Bereich von pH 5,5-6,1 liegt.
EP17208896.5A 2012-05-03 2013-04-25 Anti-il-23p19-antikörper Active EP3326649B1 (de)

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TW201840335A (zh) 2018-11-16
CN104507497B (zh) 2018-10-19
EP3326649A1 (de) 2018-05-30
CA2871985A1 (en) 2013-11-07
NZ700802A (en) 2017-06-30
AR090923A1 (es) 2014-12-17
EP2844284A1 (de) 2015-03-11
JP2015517465A (ja) 2015-06-22
CL2014002954A1 (es) 2015-02-06
IL264558A (en) 2019-02-28
US20210317201A1 (en) 2021-10-14
IL235059A0 (en) 2014-12-31
KR20150013651A (ko) 2015-02-05
TW201406391A (zh) 2014-02-16
MX2014013149A (es) 2015-01-19
CN104507497A (zh) 2015-04-08

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